Methods of inducing responsiveness to anti-angiogenic agent

ABSTRACT

The invention provides methods of inducing or improving responsiveness to a VEGF antagonist to a subject or a subject population comprising administering an adenovirus comprising a nucleic acid construct comprising a F AS-chimera gene operably linked to an endothelial cell-specific promoter and administering the VEGF antagonist.

BACKGROUND OF THE DISCLOSURE

Angiogenesis is a common and major feature of several pathologies. Amongthese are diseases in which the angiogenesis can improve the diseasecondition (such as ischemic heart disease) and diseases in which theexcessive angiogenesis is a part of the pathology and thus should beeliminated. These latter diseases include diabetes (diabeticretinopathy), cardiovascular diseases (atherosclerosis), chronicinflammation (rheumatoid arthritis), and cancer. Angiogenesis occurs intumors and permits their growth, invasion and metastasis. In 1971,Folkman proposed that tumor growth and metastases are angiogenesisdependent, and thus inhibiting angiogenesis may be a strategy to arresttumor growth.

There are several molecules involved in angiogenesis, from transcriptionfactors to growth factors. Hypoxia is an important environmental factorthat leads to neovascularization, and it induces release of severalcytokines release that are pro-angiogenic factors. Among them arevascular endothelial growth factors (VEGF) and their receptors, membersof the angiopoietin family, basic fibroblast growth factor, andendothelin-1 (ET-1). These factors are involved in induction ofangiogenesis through activation, proliferation and migration ofendothelial cells.

Recombinant forms of endogenous inhibitors of angiogenesis were testedfor the treatment of cancer. The potential pharmacokinetic,biotechnological and economic drawbacks of chronic delivery of theserecombinant inhibitors have led scientists to develop other approaches.

The development of the anti-VEGF monoclonal antibody bevacizumab hasvalidated an antiangiogenic approach as a complementary therapeuticmodality to chemotherapy. Several small molecule inhibitors, includingsecond-generation multi-targeted tyrosine kinase inhibitors, have alsoshown promise as antiangiogenic agents for cancer.

However, the potential pharmacokinetic and economic drawbacks of chronicdelivery of recombinant inhibitors, antibodies, and small molecules, aswell as the limited activity manifested when applied as monotherapy haveled scientists to evaluate antiangiogenic gene therapy. Gene therapy isan emerging modality for treating inherited and acquired human diseases.However, there are a number of obstacles limiting successful genetherapy, including duration of expression, induction of the immuneresponse, cytotoxicity of the vectors and tissue specificity. Twogeneral strategies for the cancer gene therapy were proposed: tumordirected or systemic gene therapy. The lack of success in targeting genetherapy products to cancerous cells or their environment by systemictreatments caused most therapies to be administered to the tumor itself.

BRIEF SUMMARY OF DISCLOSURE

The present invention is directed to a method for inducing or improvingresponsiveness to a vascular endothelial growth factor (VEGF) antagonistin a subject in need thereof, comprising administering a vector whichcomprises a Fas-chimera gene operably linked to an endothelial cellspecific promoter, and the VEGF antagonist to the subject, wherein theresponsiveness to the VEGF antagonist is induced or improved after theadministration of the vector compared to the responsiveness to the VEGFantagonist without the administration of the vector. The invention isfurther directed to a method for inducing or improving responsiveness toa VEGF antagonist in a subject population in need thereof, comprisingadministering a vector which comprises a Fas-chimera gene operablylinked to an endothelial cell specific promoter and the VEGF antagonistto the subject, wherein the responsiveness to the VEGF antagonist isinduced or improved after the administration of the vector compared tothe responsiveness to the VEGF antagonist without the administration ofthe vector.

In one aspect, the subject or the subject population is in need ofinducing or improving responsiveness to a VEGF antagonist. In oneembodiment, the responsiveness to the VEGF antagonist is ananti-angiogenesis property of the VEGF antagonist. In anotherembodiment, the responsiveness to the VEGF antagonist is an anti-tumoreffect of the VEGF antagonist.

In another aspect, the VEGF antagonist is an anti-VEGF antibody or aVEGF binding molecule. In one aspect of the present invention, the VEGFantagonist is bevacizumab.

In some aspects, the vector comprising a Fas-chimera gene encodes apolypeptide comprising an extracellular domain of a TNF Receptor 1(TNFR1) polypeptide fused to a transmembrane domain and an intracellulardomain of a Fas polypeptide.

One feature of the present invention is that the vector and the VEGFantagonist are administered concurrently or sequentially. Anotherfeature of the invention is that the vector is administered prior to theVEGF antagonist.

In one embodiment, the vector is administered at an effective amount ofequal to or less than about 1×10¹³, 9×10¹², 8×10¹², 7×10¹², 6×10¹²,5×10¹², 4×10¹², 3×10¹², 2×10¹², 1×10¹², 9×10¹¹, 8×10¹¹, 7×10¹¹, 6×10¹¹,5×10¹¹, 4×10¹¹, 3×10¹¹, 2×10¹¹, 1×10¹¹, 9×10¹⁰, 8×10¹⁰, 7×10¹⁰, 6×10¹⁰,5×10¹⁰, 4×10¹⁰, 3×10¹⁰, 2×10¹⁰, or 1×10¹⁰ virus particles.

In one embodiment, the vector is administered at an effective amount ofat least about 1×10¹¹ virus particles. In another embodiment, the vectoris administered at an effective amount of at least about 1×10¹² virusparticles. In another embodiment, the vector is administered at aneffective amount of at least about 1×10¹³ virus particles. In anotherembodiment, the vector is administered at an effective amount of atleast about 1×10¹⁴ virus particles.

In another embodiment of the present invention, bevacizumab isadministered at an effective amount of equal to or less than about 15mg/kg, 14 mg/kg, 13 mg/kg, 12 mg/kg, 11 mg/kg, 10 mg/kg, 9 mg/kg, 8mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg.

In another embodiment of the present invention, the vector isadministered at an effective amount of 3×10¹² to 1×10¹³ virus particlesand bevacizumab is administered at an effective amount of 5 mg/kg to 15mg/kg.

In other embodiments, the VEGF antagonist is an anti-VEGF receptorbinding antibody or a VEGF receptor binding molecule. In anotherembodiment, the anti-VEGF receptor binding antibody is a monoclonalantibody, a humanized antibody, a human antibody, a single chainantibody, or a chimeric antibody. For example, a VEGF antagonist can beselected from bevacizumab, ranibizumab, VGX-100, r84, aflibercept,IMC-18F1, IMC-1C11, ramucirumab, and any combination thereof.

In some embodiments, the vector is repeatedly administered. The vectorcan be repeatedly administered every day, once in about 2 days, once inabout 3 days, once in about 4 days, once in about 5 days, once in about6 days, once in about 7 days, once in about 2 weeks, once in about 3weeks, once in about 4 weeks, once in about 5 weeks, once in about 6weeks, once in about 7 weeks, once in about 2 months, or once in about 6months. In certain embodiments, the bevacizumab is repeatedlyadministered. In another embodiment of the invention, the bevacizumab isrepeatedly administered once in about 7 days, once in about 2 weeks,once in about 3 weeks, once in about 4 weeks, once in about 2 months,once in about 3 months, once in about 4 months, once in about 5 months,or once in about 6 months.

EMBODIMENTS

E1. A method of inducing apoptosis of an endothelial cell in a tumor ofa subject in need thereof comprising administering a VEGF antagonist tothe subject, wherein the subject is administered with a vector whichcomprises a Fas-chimera gene operably linked to an endothelial cellspecific promoter prior to the administration of the VEGF antagonist andwherein the responsiveness of the subject to the VEGF antagonist isincreased after administration of the vector.

E2. A method of inducing apoptosis of an endothelial cell in a tumor ofa subject in need thereof comprising:

-   (i) administering a vector which comprises a Fas-chimera gene    operably linked to an endothelial cell specific promoter, wherein    the responsiveness of the subject to a VEGF antagonist is increased    after administration of the vector, and-   (ii) administering the VEGF antagonist to the subject.

E3. A method of inducing apoptosis of an endothelial cell in a tumor ofa subject in need thereof comprising

-   (i) administering a vector which comprises a Fas-chimera gene    operably linked to an endothelial cell specific promoter,-   (ii) administering a VEGF antagonist to the subject, and-   (iii) measuring responsiveness of the subject to the VEGF    antagonist, wherein the responsiveness of the subject is increased    after the administration of the vector.

E4. The method of any one of embodiments E1 to E3, wherein the tumorsize of the subject is reduced after the administration of the VEGFantagonist.

E5. A method of reducing the size of a tumor in a subject in needthereof comprising administering a VEGF antagonist to the subject,wherein the subject is administered with a vector which comprises aFas-chimera gene operably linked to an endothelial cell specificpromoter prior to the administration of the VEGF antagonist, wherein theresponsiveness of the subject to the VEGF antagonist is increased afteradministration of the vector and before administration of the VEGFantagonist, and wherein the tumor size in the subject is reduced afteradministration of the VEGF antagonist.

E6. A method of reducing the size of a tumor in a subject in needthereof comprising:

-   (i) administering a vector which comprises a Fas-chimera gene    operably linked to an endothelial cell specific promoter, wherein    the responsiveness of the subject to a VEGF antagonist is increased    after the administration of the vector, and-   (ii) administering the VEGF antagonist to the subject, wherein the    tumor size in the subject is reduced after administration of the    VEGF antagonist.

E7. A method of reducing the size of a tumor in a subject in needthereof comprising:

-   (i) administering a vector which comprises a Fas-chimera gene    operably linked to an endothelial cell specific promoter,-   (ii) administering a VEGF antagonist to the subject; and-   (iii) measuring the responsiveness of the subject to the VEGF    antagonist, wherein the responsiveness of the subject to the VEGF    antagonist is increased after the administration of the vector.

E8. The method of any one of embodiments E4 to E7, wherein the tumorsize is reduced compared to the tumor size of a subject who is notadministered with the vector prior to the administration of the VEGFantagonist.

E9. The method of any one of embodiments E4 to E8, wherein the tumorsize is measured by comparing the size of the tumor prior to theadministration of the VEGF antagonist and the size of the tumor afterthe administration of the VEGF antagonist.

E10. A method of treating a disease or condition associated with tumorin a subject comprising administering a VEGF antagonist to a subject inneed thereof, wherein the subject is administered with a vector whichcomprises a Fas-chimera gene operably linked to an endothelial cellspecific promoter prior to the administration of the VEGF antagonist andwherein the responsiveness of the subject to the VEGF antagonist isincreased after administration of the vector and before administrationof the VEGF antagonist.

E11. A method of treating a disease or condition associated with tumorin a subject comprising

-   (i) administering a vector which comprises a Fas-chimera gene    operably linked to an endothelial cell specific promoter, wherein    the responsiveness of the subject to a VEGF antagonist is increased    after the administration of the vector, and-   (ii) administering the VEGF antagonist to the subject.

E12. A method of treating a disease or condition associated with tumorin a subject comprising

-   (i) administering a vector which comprises a Fas-chimera gene    operably linked to an endothelial cell specific promoter,-   (ii) administering a VEGF antagonist to the subject; and-   (iii) measuring responsiveness of the subject to the VEGF    antagonist, wherein the responsiveness of the subject to the VEGF    antagonist is increased after administration of the vector.

E13. The method of any one of embodiments E1 to E12, wherein the tumorof the subject is progressed after the administration of the vector.

E14. A method of identifying a candidate for a VEGF antagonist therapycomprising (i) measuring a tumor size of a subject who is diagnosed ashaving a tumor prior to administering a vector which comprises aFas-chimera gene operably linked to an endothelial cell specificpromoter to the subject and (ii) measuring progression of the tumorafter the administration of the vector, wherein the subject isidentified as a candidate after the tumor is progressed.

E15. A method of identifying a candidate for a VEGF antagonist therapycomprising (i) measuring a tumor size of a subject who is diagnosed ashaving a tumor prior to administering a vector which comprises aFas-chimera gene operably linked to an endothelial cell specificpromoter to the subject, (ii) measuring progression of the tumor afterthe administration of the vector, wherein the subject is identified as acandidate after the tumor is progressed, and (iii) instructing ahealthcare provider to administer a VEGF antagonist to the subject.

E16. The method of any one of embodiments E13 to E15, wherein the tumorprogression is measured by the growth of the tumor.

E17. The method of embodiment E16, wherein the growth of the tumor ismeasured by MRI.

E18. The method of any one of embodiments E1 to E17, wherein the tumorof the subject is a recurrent tumor that arose during treatment with thevector.

E19. The method of any one of embodiments E1 to E18, wherein the tumorof the subject is a metastatic tumor that arose during treatment withthe vector.

E20. The method of any one of embodiments E13-E19, wherein the tumorprogression is measured at least three weeks, at least four weeks, atleast one month, at least five weeks, at least six weeks, at least sevenweeks, at least eight weeks, at least nine weeks, at least ten weeks, atleast 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks,at least 15 weeks, or at least 16 weeks after administration of thevector.

E21. The method of any one of embodiments E1 to E20, wherein the tumorsize is measured at least one week, at least two weeks, at least threeweeks, at least four weeks, at least five weeks, at least six weeks, atleast seven weeks, or at least eight weeks prior to administration ofthe vector.

E22. The method of any one of embodiments E1 to E21, wherein the VEGFantagonist is repeatedly administered after identifying that the tumorof the subject is progressed.

E23. The method of embodiment E22, wherein the VEGF antagonist isrepeatedly administered once in about 7 days, once in about ten days,once in about 2 weeks, once in about 3 weeks, once in about 4 weeks,once in about 2 months, once in about 3 months, once in about 4 months,once in about 5 months, or once in about 6 months.

E24. The method of embodiment E22, wherein the VEGF antagonist isrepeatedly administered once in about two weeks.

E25. The method of any one of embodiments E1 to E24, wherein the vectoris repeatedly administered after administering the VEGF antagonist.

E26. The method of embodiment E25, wherein the vector is repeatedlyadministered every day, once in about 2 days, once in about 3 days, oncein about 4 days, once in about 5 days, once in about 6 days, once inabout 7 days, once in about 2 weeks, once in about 3 weeks, once inabout 4 weeks, once in about 5 weeks, once in about 6 weeks, once inabout 7 weeks, once in about 8 weeks, once in about 9 weeks, once inabout 2 months, once in about 3 months, once in about 4 months, once inabout 5 months, or once in about 6 months.

E27. The method of any one of embodiments E1 to E26, wherein the vectoris administered every 2 months and bevacizumab is administered every 2weeks.

E28. The method of any one of embodiments E1 to E27, wherein the subjectis in need of induced responsiveness to a VEGF antagonist.

E29. The method of any one of embodiments E1 to E27, wherein the subjectis in need of improved responsiveness to a VEGF antagonist.

E30. The method of any one of embodiments E1 to E29, wherein theresponsiveness of the subject to the VEGF antagonist is ananti-angiogenesis property of the VEGF antagonist.

E31. The method of any one of embodiments E1 to E30, wherein theresponsiveness of the subject to the VEGF antagonist is an anti-tumoreffect of the VEGF antagonist.

E32. The method of any one of embodiments E1 to E31, wherein the VEGFantagonist is an anti-VEGF antibody or a VEGF binding molecule.

E33. The method of embodiment E32, wherein the anti-VEGF antibody is amonoclonal antibody, a humanized antibody, a human antibody, a singlechain antibody, or a chimeric antibody.

E34. The method of any one of embodiments E1 to E33, wherein the VEGFantagonist comprises Fab, F(ab)2, Fv, or scFv.

E35. The method of any one of embodiments E1 to E34, wherein the VEGFantagonist is an anti-VEGF receptor binding antibody or a VEGF receptorbinding molecule.

E36. The method of any one of embodiments E1 to E35, wherein the VEGFantagonist is selected from bevacizumab, ranibizumab, VGX-100, r84,aflibercept, IMC-18F1, IMC-1C11, ramucirumab, and any combinationthereof.

E37. The method of embodiment E36, wherein the VEGF antagonist isbevacizumab.

E38. The method of any one of embodiments E1 to E37, wherein theFas-chimera gene product comprises a polypeptide comprising anextracellular domain of a TNF Receptor 1 (TNFR1) polypeptide fused to atransmembrane domain and an intracellular domain of a Fas polypeptide.

E39. The method of embodiment E38, wherein the extracellular domain ofthe TNFR1 comprises an amino acid sequence at least 70%, 80%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4, wherein theextracellular domain of the TNFR1 is capable of binding to TNF-α.

E40. The method of embodiment E39, wherein the trans-membrane domain andthe intracellular domain of the Fas polypeptide comprises an amino acidsequence at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 8, wherein the trans-membrane domain and theintracellular domain of the Fas polypeptide is capable of inducing Fasmediated apoptosis.

E41. The method of any one of embodiments E1 to E40, wherein theFas-chimera gene comprises a first nucleotide sequence, which is atleast 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQID NO: 3, and a second nucleotide sequence, which is at least 70%, 80%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

E42. The method of any one of embodiments E1 to E41, wherein theendothelial cell specific promoter comprises a PPE-1 promoter.

E43. The method of any one of embodiments E1 to E42, wherein theendothelial cell specific promoter further comprises an enhancer.

E44. The method of embodiment E43, wherein the enhancer comprises anucleotide sequence at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to SEQ ID NO: 15 or SEQ ID NO: 16, wherein the enhancerinduces an improved endothelial cell specificity compared to anendothelial cell specific promoter without the enhancer.

E45. The method of embodiment E44, wherein the enhancer comprises SEQ IDNO: 11 or SEQ ID NO: 12.

E46. The method of embodiment E45, wherein the enhancer furthercomprises SEQ ID NO: 13 or SEQ ID NO: 14.

E47. The method of any one of embodiments E1 to E46, wherein theendothelial cell specific promoter is a PPE-1-3× promoter.

E48. The method of embodiment E47, wherein the PPE-1-3× promotercomprises a nucleotide sequence at least 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 18, wherein the PPE-1-3×promoter is capable of directing the Fas-chimera gene expression inendothelial cells.

E49. The method of any one of embodiments E1 to E48, wherein the vectordoes not contain an E1 region of an adenovirus.

E50. The method of any one of embodiments E1 to E49, wherein the vectorand the VEGF antagonist are administered concurrently or sequentially.

E51. The method of embodiment E50, wherein the vector is administeredprior to the VEGF antagonist.

E52. The method of any one of embodiments E1 to E51, wherein the vectoris administered at an effective amount of equal to or less than about1×10¹³, 9×10¹², 8×10¹², 7×10¹², 6×10¹², 5×10¹², 4×10¹², 3×10¹², 2×10¹²,1×10¹², 9×10″, 8×10″, 7×10¹¹, 6×10″, 5×10¹¹, 4×10¹¹, 3×10¹¹, 2×10¹¹,1×10¹¹, 9×10¹⁰, 8×10¹⁰, 7×10¹⁰, 6×10¹⁰, 5×10¹⁰, 4×10¹⁰, 3×10¹⁰, 2×10¹⁰,or 1×10¹⁰ virus particles.

E53. The method of any one of embodiments E36 to E52, whereinbevacizumab is administered at an effective amount of less than about 15mg/kg, 14 mg/kg, 13 mg/kg, 12 mg/kg, 11 mg/kg, 10 mg/kg, 9 mg/kg, 8mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg.

E54. The method of any one of embodiments E36 to E53, wherein the vectoris administered at an effective amount of 3×10¹² to 1 to 10¹³ virusparticles and bevacizumab is administered at an effective amount of 5mg/kg to 15 mg/kg.

E55. The method of any one of embodiments E32 to E54, wherein theanti-VEGF antibody or the VEGF binding molecule comprises at least oneCDR selected from the group consisting of V_(H) CDR1 (SEQ ID NO: 28),V_(H) CDR2 (SEQ ID NO: 29), V_(H) CDR3 (SEQ ID NO: 30), V_(L) CDR1 (SEQID NO: 31), V_(H) CDR2 (SEQ ID NO: 32), V_(H) CDR3 (SEQ ID NO: 33), andany combination thereof.

E56. The method of embodiment E55, wherein the anti-VEGF antibody or theVEGF binding molecule comprises V_(H) CDR1 (SEQ ID NO: 28), V_(H) CDR2(SEQ ID NO: 29), and V_(H) CDR3 (SEQ ID NO: 30).

E57. The method of embodiment E55, wherein the anti-VEGF antibody or theVEGF binding molecule comprises V_(L) CDR1 (SEQ ID NO: 31), V_(L) CDR2(SEQ ID NO: 32), and V_(L) CDR3 (SEQ ID NO: 33).

E58. The method of embodiment E55, wherein the anti-VEGF antibody orVEGF binding molecule comprises V_(H) CDR1 (SEQ ID NO: 28), V_(H) CDR2(SEQ ID NO: 29), V_(H) CDR3 (SEQ ID NO: 30), V_(L) CDR1 (SEQ ID NO: 31),V_(L) CDR2 (SEQ ID NO: 32), and V_(L) CDR3 (SEQ ID NO: 33).

E59. The method of any one of embodiments E1 to E58, wherein the subjectis in need of a reduction of angiogenesis.

E60. The method of embodiment E59, wherein the vector and the VEGFantagonist reduce angiogenesis.

E61. The method of any one of embodiments E1 to E60, wherein the vectoris an adenovirus vector.

E62. The method of embodiment E61, wherein the adenovirus vector isadenovirus serotype 5.

E63. The method of any one of embodiments E1 to E62, wherein the vectorcomprises, consists of, or consists essentially of SEQ ID NO: 19.

E64. The method of any one of embodiments E1 to E63, wherein the vectoris an isolated virus having European Collection of Cell Cultures (ECACC)Accession Number 13021201.

E65. The method of any one of embodiments E1 to E64, wherein thepromoter comprises a hypoxia response element.

E66. The method of any one of embodiments E1 to E65, wherein the vectoris administered at an effective amount of about 1×10¹¹, about 1×10¹²,about 1×10¹³, or about 1×10¹⁴ virus particles.

E67. The method of any one of embodiments E1 to E66, wherein the tumoris a brain tumor.

E68. The method of embodiment E67, wherein the brain tumor isglioblastoma multiforme.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows a combination therapy regimen of an adenovirus comprising aFAS-chimera gene operably linked to an endothelial cell-specificpromoter (e.g., VB-111) and bevacizumab (BEV). 1×10¹³ VPs of VB-111 canbe administered every eight weeks, and bevacizumab can be administeredevery two weeks. MRI assessment can be performed every eight weeks toassess the tumor progression.

FIG. 2 shows the time for glioblastima disease progression from firstrelapse to second relapse in patients receiving a combination therapyregimen of an adenovirus comprising a FAS-chimera gene operably linkedto an endothelial cell-specific promoter (e.g., VB-111) and bevacizumab(BEV).

FIG. 3 shows an example of a patient who was treated with a combinationregimen of an adenovirus comprising a FAS-chimera gene operably linkedto an endothelial cell-specific promoter (e.g., VB-111) and bevacizumab(BEV) upon glioblastoma disease progression.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application including the definitions will control. Unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. All publications, patentsand other references mentioned herein are incorporated by reference intheir entireties for all purposes as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although methods and materials similar or equivalent to those describedherein can be used in practice or testing of the present invention,suitable methods and materials are described below. The materials,methods and examples are illustrative only and are not intended to belimiting. Other features and advantages of the invention will beapparent from the detailed description and from the claims.

In order to further define this invention, the following terms anddefinitions are provided.

The term “about” is used herein to mean approximately, roughly, around,or in the regions of. When the term “about” is used in conjunction witha numerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10 percent, up or down (higher or lower).

As used herein, “antibody” means an intact immunoglobulin, anantigen-binding fragment thereof, or an antigen-binding molecule.Antibodies of this invention can be of any isotype or class (e.g., M, D,G, E and A) or any subclass (e.g., G1-4, A1-2) and can have either akappa (κ) or lambda (λ) light chain.

The term “effective amount” as used herein refers to an amounteffective, at dosages and for periods of time necessary, to achieve adesired result. A desired result can be, for example, reduction orinhibition of neo-vascularization or angiogenesis in vitro or in vivo.An effective amount need not be a complete removal ofneo-vascularization or angiogenesis.

As used herein, a “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve adesired therapeutic result. A desired therapeutic result may be, e.g.,lessening of symptoms, regression or stabilization of tumor size inradiological imaging, prolonged survival, improved mobility, and thelike. A therapeutic result need not be a “cure.” In some embodiments, atherapeutically effective amount is an amount or dosage that isnecessary to prevent occurrence of a tumor.

As used herein, a “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result. In some embodiments, since aprophylactic dose is used in subjects prior to or at an earlier stage ofdisease, the prophylactically effective amount will be less than thetherapeutically effective amount.

The term “polynucleotide” or “nucleotide” is intended to encompass asingular nucleic acid as well as plural nucleic acids, and refers to anisolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA)or plasmid DNA (pDNA). In certain embodiments, a polynucleotidecomprises a conventional phosphodiester bond or a non-conventional bond(e.g., an amide bond, such as found in peptide nucleic acids (PNA)).

As used herein, a “polynucleotide,” “nucleotide,” or “nucleic acid” canbe used interchangeably and contain the nucleotide sequence of thefull-length cDNA sequence, including the untranslated 5′ and 3′sequences, the coding sequences, as well as fragments, epitopes,domains, and variants of the nucleic acid sequence. The polynucleotidecan be composed of any polyribonucleotide or polydeoxyribonucleotide,which may be unmodified RNA or DNA or modified RNA or DNA. For example,polynucleotides can be composed of single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, the polynucleotidescan be composed of triple-stranded regions comprising RNA or DNA or bothRNA and DNA. Polynucleotides may also contain one or more modified basesor DNA or RNA backbones modified for stability or for other reasons.“Modified” bases include, for example, tritylated bases and unusualbases such as inosine. A variety of modifications can be made to DNA andRNA; thus, “polynucleotide” embraces chemically, enzymatically, ormetabolically modified forms.

In the present invention, a polypeptide can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids (e.g. non-naturally occurring amino acids). Thepolypeptides of the present invention may be modified by either naturalprocess, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also, a given polypeptide may contain many types ofmodifications. Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, Proteins—Structure And Molecular Properties, 2nd Ed., T. E.Creighton, W.H. Freeman and Company, New York (1993); PosttranslationalCovalent Modification of Proteins, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

The terms “fragment,” “variant,” “derivative” and “analog” whenreferring to any polypeptide or polynucleotide of the present inventioninclude any polypeptides or polynucleotides which retain at least someactivities, i.e., the ability to function as any naturally-occurringfunction of the polypeptide or polynucleotide. For example, a“fragment,” “variant,” “derivative” and “analog” of Tumor necrosisfactor Receptor 1 (TNFR1) has some activities of the naturally occurringfull-length TNFR1, e.g., the ability to bind to TNFR1 ligand, i.e.,TNF-alpha or lymphotoxin. In another example, a “fragment,” “variant,”“derivative” and “analog” of a FAS polypeptide have some activities of anaturally-occurring full-length FAS polypeptide, e.g., the ability toinduce apoptosis. In other examples, a “fragment,” “variant,”“derivative” and “analog” of an endothelial cell-specific promoter caninduce endothelial cell-specific expression of a gene operably linked tothe promoter. Additional non-limiting examples of the various fragments,variants, analogues, or derivatives of the TNFR1, FAS polypeptide, andendothelial cell-specific promoters are described below.

In the present invention, a “polypeptide fragment” or “protein fragment”refers to a short amino acid sequence of a polypeptide. Protein orpolypeptide fragments may be “free-standing,” or comprised within alarger polypeptide of which the fragment forms a part of region.Representative examples of polypeptide fragments of the invention,include, for example, fragments comprising about 5 amino acids, about 10amino acids, about 15 amino acids, about 20 amino acids, about 30 aminoacids, about 40 amino acids, about 50 amino acids, about 60 amino acids,about 70 amino acids, about 80 amino acids, about 90 amino acids, orabout 100 amino acids.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, if an amino acid in apolypeptide is replaced with another amino acid from the same side chainfamily, the substitution is considered to be conservative. In anotherembodiment, a string of amino acids can be conservatively replaced witha structurally similar string that differs in order and/or compositionof side chain family members.

The term “percent sequence identity” between two polynucleotide orpolypeptide sequences refers to the number of identical matchedpositions shared by the sequences over a comparison window, taking intoaccount additions or deletions (i.e., gaps) that must be introduced foroptimal alignment of the two sequences. A matched position is anyposition where an identical nucleotide or amino acid is presented inboth the target and reference sequence. Gaps presented in the targetsequence are not counted since gaps are not nucleotides or amino acids.Likewise, gaps presented in the reference sequence are not counted sincetarget sequence nucleotides or amino acids are counted, not nucleotidesor amino acids from the reference sequence.

The percentage of sequence identity is calculated by determining thenumber of positions at which the identical amino-acid residue or nucleicacid base occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. The comparison ofsequences and determination of percent sequence identity between twosequences may be accomplished using readily available software both foronline use and for download. Suitable software programs are availablefrom various sources, and for alignment of both protein and nucleotidesequences. One suitable program to determine percent sequence identityis bl2seq, part of the BLAST suite of program available from the U.S.government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq performs a comparison between twosequences using either the BLASTN or BLASTP algorithm. BLASTN is used tocompare nucleic acid sequences, while BLASTP is used to compare aminoacid sequences. Other suitable programs are, e.g., Needle, Stretcher,Water, or Matcher, part of the EMBOSS suite of bioinformatics programsand also available from the European Bioinformatics Institute (EBI) atwww.ebi.ac.uk/Tools/psa.

Different regions within a single polynucleotide or polypeptide targetsequence that aligns with a polynucleotide or polypeptide referencesequence can each have their own percent sequence identity. It is notedthat the percent sequence identity value is rounded to the nearesttenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to80.2. It also is noted that the length value will always be an integer.

One skilled in the art will appreciate that the generation of a sequencealignment for the calculation of a percent sequence identity is notlimited to binary sequence-sequence comparisons exclusively driven byprimary sequence data. Sequence alignments can be derived from multiplesequence alignments. One suitable program to generate multiple sequencealignments is ClustalW2, available from www.clustal.org. Anothersuitable program is MUSCLE, available from www.drive5.com/muscle/.ClustalW2 and MUSCLE are alternatively available, e.g., from the EBI.

It will also be appreciated that sequence alignments can be generated byintegrating sequence data with data from heterogeneous sources such asstructural data (e.g., crystallographic protein structures), functionaldata (e.g., location of mutations), or phylogenetic data. A suitableprogram that integrates heterogeneous data to generate a multiplesequence alignment is T-Coffee, available at www.tcoffee.org, andalternatively available, e.g., from the EBI. It will also be appreciatedthat the final alignment used to calculated percent sequence identitymay be curated either automatically or manually.

As used herein, the terms “linked,” “fused,” “fusion,” “chimeric,” and“chimera” are used interchangeably. These terms refer to the joiningtogether of two more elements or components, by whatever means includingchemical conjugation or recombinant means. An “in-frame fusion” refersto the joining of two or more open reading frames (ORFs) to form acontinuous longer ORF, in a manner that maintains the correct readingframe of the original ORFs. Thus, the resulting recombinant fusion orchimeric protein is a single protein containing two or more segmentsthat correspond to polypeptides encoded by the original ORFs (whichsegments are not normally so joined in nature.) Although the readingframe is thus made continuous throughout the fused segments, thesegments may be physically or spatially separated by, for example,in-frame linker sequence.

The terms “heterologous” and “heterologous moiety” mean that apolynucleotide, polypeptide, or other moiety is derived from a distinctentity from that of the entity to which it is being compared. Forinstance, a heterologous polypeptide can be synthetic, or derived from adifferent species, different cell type of an individual, or the same ordifferent type of cell of distinct individuals. In one aspect, aheterologous moiety can be a polypeptide fused to another polypeptide toproduce a fusion polypeptide or protein. In another aspect, aheterologous moiety can be a non-polypeptide such as PEG conjugated to apolypeptide or protein.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes withoutlimitation transcription of the gene into messenger RNA (mRNA), transferRNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) orany other RNA product and the translation of such mRNA intopolypeptide(s). If the final desired product is biochemical, expressionincludes the creation of that biochemical and any precursors.

The term “complementarity determining region” (CDR) as used hereinrefers to the amino acid residues of an antibody which are responsiblefor binding to an antigen. The CDR regions of an antibody are foundwithin the hypervariable region of both heavy and light chains of theantibody. Full length antibodies comprise three CDR regions in the heavychain variable domain and three CDR regions in the light chain variabledomain.

The term “repeatedly administered” as used herein refers toadministration of a therapeutic agent on a repeated basis at defined,fixed intervals. The intervals of time between each administration maybe altered during the course of the repeated administration and may beas long as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months,4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months,11 months, 1 year, or more.

The term “combination therapy” as used herein refers to theadministration of two or more therapeutic modalities to treat a diseaseor condition. In one aspect of the present invention, combinationtherapy refers to the administration of a VEGF antagonist and a vectorto a subject or a subject population in need thereof. In someembodiments, the combination therapy comprises administering the VEGFantagonist prior to administering the vector. In another embodiment, thecombination therapy comprises administering the VEGF antagonistconcomitantly with administration of the vector. In another embodiment,the combination therapy comprises administering the VEGF antagonistafter administering the vector.

II. Nucleic Acid Constructs Comprising a Fas-Chimera Gene and anEndothelial Cell Specific Promoter

The present invention is related to methods of inducing or improvingresponsiveness to a VEGF antagonist in a subject or a subject populationcomprising administering a vector and a VEGF antagonist. The geneencoding the FAS-chimera protein (or gene product), in the presentinvention can be linked to an endothelial cell-specific promoter, whichdirects expression of the FAS-chimera gene product in an endothelialcell. Expression of such a cytotoxic gene product is useful in asituation where excessive neo-vascularization or blood vessel growth isnot desirable, e.g., in a tumor.

The present invention also provides a homogeneous population of anucleic acid construct comprising a FAS-chimera gene operably linked toan endothelial cell-specific promoter.

A. FAS-Chimera

A FAS-chimera protein expressed by the nucleic acid construct of theinvention comprises at least two “death receptor” polypeptides, each ofthe polypeptides is derived from a different protein. The firstpolypeptide of the FAS-chimera protein comprises a ligand binding domainof Tumor Necrosis Factor Receptor 1 (TNFR1). The second polypeptide ofthe FAS-chimera protein comprises an effector domain of a FASpolypeptide.

The ligand binding domain of TNFR1 can be any domain that binds to aTNFR1 ligand. In one embodiment, the TNFR1 ligand is TNF-α. In anotherembodiment, the TNFR1 ligand is lymphotoxin-α. The ligand binding domainof TNFR1 can be an extracellular domain of TNFR1 or any fragments,variants, derivatives, or analogues thereof. Non-limiting examples ofthe TNFR1 ligand binding domain are described below.

The effector domain of a FAS polypeptide useful for the inventioncomprises any FAS domains that form death-inducing signaling complex(DISC), thereby inducing apoptosis. In one embodiment, an effectordomain of a FAS polypeptide comprises an intracellular domain, atrans-membrane domain, or both. Non-limiting examples of FAS polypeptideeffector domains are described below.

The TNFR1 and the FAS polypeptide can be linked by a peptide bond or bya linker. The linker connecting the TNFR1 ligand binding domain with theFAS effector domain can be a polypeptide linker or a non-peptide linker.For example, a linker for the FAS-chimera protein can comprise one ormore glycine, serine, leucine, or any combinations thereof. In oneembodiment, a linker useful for the invention comprises Ser-Leu. Inanother embodiment, a linker useful for the invention comprises (GGGS)n,(Denise et al. J. Biol. Chem. 277:35035-35043 (2002)), wherein n can be0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more (SEQ ID NO: 27).

1. Tumor Necrosis Factor Receptor 1

The full-length human TNFR1 polypeptide is 455 amino acids in length andis also known as TNF-R1, Tumor necrosis factor receptor type I (TNFRI),TNFR-I, TNFRSF1A, TNFAR, p55, P60, or CD120a. Naturally-occurring humanTNFR1 polypeptide is known to bind to TNF-α or homotrimericlymphotoxin-α. Binding of TNF-α to the extracellular domain leads tohomotrimerization of TNFR1, which then interacts specifically with thedeath domain of Tumor Necrosis Factor Receptor Type 1-Associated DeathDomain Protein (TRADD). Various TRADD-interacting proteins such as TNFReceptor Associated Factors (TRAFS), Receptor-InteractingSerine/Threonine-Protein Kinase 1 (RIPK1), and Fas-Associated Proteinwith Death Domain (FADD) are recruited to the complex by theirassociation with TRADD. The complex activates at least two distinctsignaling cascades, apoptosis and NF-kappa-B signaling.

A 455 aa polypeptide sequence reported as a human TNFR1 polypeptidesequence has the identifier number P19438-1 in the UniProtKB database.This human TNFR1 polypeptide sequence is designated herein as isoform Aand SEQ ID NO: 2. SEQ ID NO: 1 is a nucleotide sequence encoding SEQ IDNO: 2. A polypeptide sequence of 108 aa was reported as an isoform ofthe human TNFR1 polypeptide sequence and has the identifier numberP19438-2 in the UniProtKB database. The 108 aa polypeptide correspondsto amino acids 1 to 108 of isoform A (SEQ ID NO: 2) and is designatedherein as isoform B. Another variant of the human TNFR1 polypeptidehaving 232 aa was reported as the identifier number P19438-3 in theUniProtKB database. The 232 aa polypeptide corresponds to amino acids 1to 232 of isoform A (SEQ ID NO: 2) and is designated herein as isoformC. Additional natural variants of human TNFR1 include, but are notlimited to, the TNFR1 polypeptide of isoforms A, B, and C comprising oneor more mutations selected from the group consisting of H51Q, C59R,C59S, C62G, C62Y, P75L, T79M, C81F, C99S, S115G, C117R, C117Y, R121P,R121Q, P305T, and any combinations thereof. Other known TNFR1 variantsinclude the TNFR1 polypeptide of isoforms A, B, and C comprisingL13LILPQ, K255E, S286G, R394L, 412:Missing, GPAA443-446APP, or anycombinations thereof.

Table 1 shows the human wild-type TNFR1 amino acid sequence and anucleotide sequence encoding the wild-type TNFR1.

TABLE 1 TNFR1 Sequences SEQ ID No. Sequences AminoMGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCT acidKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVD sequenceRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECV of TNFR1SCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFGLCLLSLLFIGLMYRYQRWK (SEQ IDSKLYSIVCGKSTPEKEGELEGTTTKPLAPNPSFSPTPGFTPTLGFSPVPSSTFTSSSTYT NO: 2)PGDCPNFAAPRREVAPPYQGADPILATALASDPIPNPLQKWEDSAHKPQSLDTDDPATLYAVVENVPPLRWKEFVRRLGLSDHEIDRLELQNGRCLREAQYSMLATWRRRTPRREATLELLGRVLRDMDLLGCLEDIEEALCGPAALPPAPSLLR NucleotideAtgggcctctccaccgtgcctgacctgctgctgccgctggtgctcctggagctgttggtg SequenceGgaatatacccctcaggggttattggactggtccctcacctaggggacagggagaagaga encodingGatagtgtgtgtccccaaggaaaatatatccaccctcaaaataattcgatttgctgtacc TNFR1Aagtgccacaaaggaacctacttgtacaatgactgtccaggcccggggcaggatacggac (SEQ IDTgcagggagtgtgagagcggctccttcaccgcttcagaaaaccacctcagacactgcctc NO: 1)AgctgctccaaatgccgaaaggaaatgggtcaggtggagatctcttcttgcacagtggacCgggacaccgtgtgtggctgcaggaagaaccagtaccggcattattggagtgaaaaccttTtccagtgcttcaattgcagcctctgcctcaatgggaccgtgcacctctcctgccaggagAaacagaacaccgtgtgcacctgccatgcaggtttctttctaagagaaaacgagtgtgtcTcctgtagtaactgtaagaaaagcctggagtgcacgaagttgtgcctaccccagattgagAatgttaagggcactgaggactcaggcaccacagtgctgttgcccctggtcattttctttGgtctttgccttttatccctcctcttcattggtttaatgtatcgctaccaacggtggaagTccaagctctactccattgtttgtgggaaatcgacacctgaaaaagagggggagcttgaaGgaactactactaagcccctggccccaaacccaagcttcagtcccactccaggcttcaccCccaccctgggcttcagtcccgtgcccagttccaccttcacctccagctccacctataccCccggtgactgtcccaactttgcggctccccgcagagaggtggcaccaccctatcaggggGctgaccccatccttgcgacagccctcgcctccgaccccatccccaacccccttcagaagTgggaggacagcgcccacaagccacagagcctagacactgatgaccccgcgacgctgtacGccgtggtggagaacgtgcccccgttgcgctggaaggaattcgtgcggcgcctagggctgAgcgaccacgagatcgatcggctggagctgcagaacgggcgctgcctgcgcgaggcgcaaTacagcatgctggcgacctggaggcggcgcacgccgcggcgcgaggccacgctggagctgCtgggacgcgtgctccgcgacatggacctgctgggctgcctggaggacatcgaggaggcgctttgcggccccgccgccctcccgcccgcgcccagtcttctcaga AminoMGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCT acidKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVD sequenceRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECV of aSCSNCKKSLECTKLCLP Ligand Binding Domain of TNFR1 (SEQ ID NO: 4)Nucleotide atgggcctct ccaccgtgcc tgacctgctg ctgccgctgg tgctcctggasequence gctgttggtg ggaatatacc cctcaggggt tattggactg gtccctcaccencoding a  taggggacag ggagaagaga gatagtgtgt gtccccaagg aaaatatatcLigand caccctcaaa ataattcgat ttgctgtacc aagtgccaca aaggaaccta Bindingcttgtacaat gactgtccag gcccggggca ggatacggac tgcagggagt Domain ofgtgagagcgg ctccttcacc gcttcagaaa accacctcag acactgcctc TNFR1agctgctcca aatgccgaaa ggaaatgggt caggtggaga tctcttcttg (SEQ IDcacagtggac cgggacaccg tgtgtggctg caggaagaac cagtaccggc NO: 3)attattggag tgaaaacctt ttccagtgct tcaattgcag cctctgcctcaatgggaccg tgcacctctc ctgccaggag aaacagaaca ccgtgtgcacctgccatgca ggtttctttc taagagaaaa cgagtgtgtc tcctgtagtaactgtaagaa aagcctggag tgcacgaagt tgtgcctacc a

The mouse TNFR1 polypeptide sequence and its variants are also reported.The 454 aa mouse TNFR1 polypeptide has the identifier number P25118 inUniProtKB database. TNFR1 polypeptides known in other animals include,but are not limited to, rat (e.g., P22934 in the UniProtKB database),cow (e.g., 019131 in the UniProtKB database), pig (e.g., P50555 in theUniProtKB database), or horse (e.g., D1MH71 in the UniProtKB database).

The full-length TNFR1 can be cleaved into two chains, (1) TNF ReceptorSuperfamily Member 1A, membrane form (i.e., amino acids 22 to 455corresponding to full-length TNFR1) and (2) TNF-binding protein 1 (TBPI)(i.e., amino acids 41 to 291 corresponding to full-length TNFR1). Thefull-length human TNFR1 polypeptide consists of a signal sequence (aminoacids 1 to 21 of SEQ ID NO: 2), an extracellular domain (amino acids 22to 211 of SEQ ID NO: 2), a trans-membrane domain (amino acids 212 to 234of SEQ ID NO: 2), and a cytoplasmic domain (amino acids 235 to 455 ofSEQ ID NO: 2). The TNFR1 extracellular domain comprises four cysteinerepeat regions, TNFR-Cys1 (amino acids 43 to 82 corresponding to SEQ IDNO: 2), TNFR-Cys2 (amino acids 83 to 125 corresponding to SEQ ID NO: 2),TNFR-Cys3 (amino acids 126 to 166 corresponding to SEQ ID NO: 2), andTNFR-Cys4 (amino acids 167 to 196 corresponding to SEQ ID NO: 2).

As one of skill in the art will appreciate, the beginning and endingresidues of the domains listed above can vary depending upon thecomputer modeling program used or the method used for determining thedomain. As such, various functional domains of TNFR1 may vary from thosedefined above.

In one embodiment, a ligand binding domain of TNFR1 useful for theFAS-chimera protein comprises, consists essentially of, or consists ofan extracellular domain of TNFR1, or any fragment, variant, derivative,or analogue thereof, wherein the extracellular domain of TNFR1, or anyfragment, variant, derivative, or analogue thereof binds to TNF-α. Inanother embodiment, a ligand binding domain of TNFR1 comprisesTNFR-Cys1; TNFR-Cys2; TNFR-Cys3; TNFR-Cys4; TNFR-Cys1 and TNFR-Cys2;TNFR-Cys1 and TNFR-Cys3; TNFR-Cys1 and TNFR-Cys4; TNFR-Cys2 andTNFR-Cys3; TNFR-Cys2 and TNFR-Cys4; TNFR-Cys3 and TNFR-Cys4; TNFR-Cys1,TNFR-Cys2, and TNFR-Cys3; TNFR-Cys1, TNFR-Cys2, and TNFR-Cys4;TNFR-Cys2, TNFR-Cys3, and TNFR-Cys4; or TNFR-Cys1, TNFR-Cys2, TNFR-Cys3,and TNFR-Cys4. In other embodiments, a ligand binding domain of TNFR1 inthe FAS-chimera protein comprises TNF binding protein I. In yet otherembodiments, a TNFR1 ligand binding domain of the FAS-chimera proteincomprises, consists essentially of, or consists of an amino acidsequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids 22 to 190, amino acids 22 to 191, amino acids22 to 192, amino acids 22 to 193, amino acids 22 to 194, amino acids 22to 195, amino acids 22 to 196, amino acids 22 to 197, amino acids 22 to198, amino acids 22 to 199, amino acids 22 to 200, amino acids 22 to201, amino acids 22 to 202, amino acids 22 to 203, amino acids 22 to204, amino acids 22 to 205, amino acids 22 to 206, amino acids 22 to207, amino acids 22 to 208, amino acids 22 to 209, amino acids 22 to210, or amino acids 22 to 211 of SEQ ID NO: 2, wherein the ligandbinding domain binds to a TNFR1 ligand, e.g., TNF-α.

In other embodiments, the ligand binding domain of TNFR1 furthercomprises a signal peptide. One example of the suitable signal peptidesis the signal peptide of TNFR1, e.g., amino acids 1 to 21 of SEQ ID NO:2. In yet other embodiments, a ligand binding domain of the FAS-chimeragene product comprises, consists essentially of, or consists of an aminoacid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids 1 to 190, amino acids 1 to 191, aminoacids 1 to 192, amino acids 1 to 193, amino acids 1 to 194, amino acids1 to 195, amino acids 1 to 196, amino acids 1 to 197, amino acids 1 to198, amino acids 1 to 199, amino acids 1 to 200, amino acids 1 to 201,amino acids 1 to 202, amino acids 1 to 203, amino acids 1 to 204, aminoacids 1 to 205, amino acids 1 to 206, amino acids 1 to 207, amino acids1 to 208, amino acids 1 to 209, amino acids 1 to 210, or amino acids 1to 211 of SEQ ID NO: 2, wherein the ligand binding domain binds to aTNFR1 ligand, e.g., TNF-α. In a specific embodiment, a TNFR1 ligandbinding domain of the FAS-chimera protein comprises, consistsessentially of, or consists of an amino acid sequence at least 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4,wherein the ligand binding domain binds to a TNFR1 ligand, e.g., TNF-α.

In yet other embodiments, the ligand binding domain of TNFR1 is encodedby a nucleotide sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 3.

In still other embodiments, a TNFR1 ligand binding domain of theFAS-chimera protein comprises, consists essentially of, or consists ofan amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to amino acids 22 to 108 of SEQ ID NO: 2 (TNFR1isoform B), amino acids 22 to 232 of SEQ ID NO: 2 (TNFR1 isoform C), oramino acids 44 to 291 of SEQ ID NO: 2 (TBP1), wherein the ligand bindingdomain binds to a TNFR1 ligand, e.g., TNF-α.

2. FAS Polypeptide

The full-length human FAS polypeptide is 335 amino acids in length andis also known as Tumor Necrosis Factor Receptor Superfamily Member 6,Apo-1 antigen, Apoptosis-mediating surface antigen FAS, FASLG receptor,or CD95. Naturally occurring FAS polypeptide is a receptor forTNFSF6/FASLG. When the FAS polypeptide binds to the FAS ligand (FasL),the interaction between FAS and FasL results in the formation of thedeath-inducing signaling complex (DISC), which contains the FADD,caspase-8 and caspase-10. In some types of cells (type I), processedcaspase-8 directly activates other members of the caspase family, andtriggers the execution of apoptosis of the cell. In other types of cells(type II), the FAS-DISC starts a feedback loop that spirals intoincreasing release of proapoptotic factors from mitochondria and theamplified activation of caspase-8. FAS-mediated apoptosis may have arole in the induction of peripheral tolerance, in the antigen-stimulatedsuicide of mature cells or both.

A 335 aa polypeptide sequence reported as a human FAS polypeptidesequence has the identifier number P25445-1 in the UniProtKB database.This human FAS polypeptide sequence is designated herein as SEQ ID NO:6. SEQ ID NO: 5 is a nucleotide sequence encoding SEQ ID NO: 6. Thenucleotide sequence encoding the FAS polypeptide is also known as APT1,FAST, or TNFRSF6. The full-length FAS polypeptide contains a signalpeptide (amino acids 1 to 25 corresponding to SEQ ID NO: 6), anextracellular domain (amino acids 26 to 173 corresponding to SEQ ID NO:6), a trans-membrane domain (amino acids 174 to 190 corresponding to SEQID NO: 6), and an intracellular (or cytoplasmic) domain (amino acids 191to 335 corresponding to SEQ ID NO: 6). The intracellular domain containsa death domain (e.g., amino acids 230 to 314 corresponding to SEQ ID NO:6).

As one of skill in the art will appreciate, the beginning and endingresidues of the domains listed above may vary depending upon thecomputer modeling program used or the method used for determining thedomain. As such, various functional domains of FAS may vary from thosedefined above. Table 2 shows the wild-type human FAS amino acid sequenceand a nucleotide sequence encoding the FAS protein.

TABLE 2 FAS Sequences Sequences Amino acidMLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCH sequence ofKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCT human FASRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSNLGWLCLL proteinLLPIPLIVWVKRKEVQKTCRKHRKENQGSHESPTLNPETVAINLSDVDLSKYITTIAGVM (SEQ IDTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKK NO: 6)ANLCTLAEKIQTIILKDITSDSENSNFRNEIQSLV NucleotideAtgctgggcatctggaccctcctacctctggttcttacgtctgttgctagattatcgtcc sequenceAaaagtgttaatgcccaagtgactgacatcaactccaagggattggaattgaggaagact encodingGttactacagttgagactcagaacttggaaggcctgcatcatgatggccaattctgccat human FASAagccctgtcctccaggtgaaaggaaagctagggactgcacagtcaatggggatgaacca sequenceGactgcgtgccctgccaagaagggaaggagtacacagacaaagcccatttttcttccaaa (SEQ IDTgcagaagatgtagattgtgtgatgaaggacatggcttagaagtggaaataaactgcacc NO: 5)CggacccagaataccaagtgcagatgtaaaccaaactttttttgtaactctactgtatgtGaacactgtgacccttgcaccaaatgtgaacatggaatcatcaaggaatgcacactcaccAgcaacaccaagtgcaaagaggaaggatccagatctaacttggggtggctttgtcttcttCttttgccaattccactaattgtttgggtgaagagaaaggaagtacagaaaacatgcagaAagcacagaaaggaaaaccaaggttctcatgaatctccaactttaaatcctgaaacagtgGcaataaatttatctgatgttgacttgagtaaatatatcaccactattgctggagtcatgAcactaagtcaagttaaaggctttgttcgaaagaatggtgtcaatgaagccaaaatagatGagatcaagaatgacaatgtccaagacacagcagaacagaaagttcaactgcttcgtaatTggcatcaacttcatggaaagaaagaagcgtatgacacattgattaaagatctcaaaaaaGccaatctttgtactcttgcagagaaaattcagactatcatcctcaaggacattactagtGactcagaaaattcaaacttcagaaatgaaatccaaagcttggtctag Amino acidGSRSNLGWLCLLLLPIPLIVWVKRKEVQKTCRKHRKENQGS sequence ofHESPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVR an EffectorKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAY Domain ofDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEIQ FAS (SEQ SLV ID NO: 8)Nucleotide Aggatccagatctaacttggggtggctttgtcttcttcttttgccaattccactaattsequence Gtttgggtgaagagaaaggaagtacagaaaacatgcagaaagcacagaaaggaaaaccencoding an AaggttctcatgaatctccaaccttaaatcctgaaacagtggcaataaatttatctgaEffector TgttgacttgagtaaatatatcaccactattgctggagtcatgacactaagtcaagttDomain of AaaggctttgttcgaaagaatggtgtcaatgaagccaaaatagatgagatcaagaatgFAS (SEQ AcaatgtccaagacacagcagaacagaaagttcaactgcttcgtaattggcatcaactID NO: 7) TcatggaaagaaagaagcgtatgacacattgattaaagatctcaaaaaagccaatcttTgtactcttgcagagaaaattcagactatcatcctcaaggacattactagtgactcagaaaattcaaacttcagaaatgaaatccaaagcttggtctag

The mouse FAS polypeptide sequence and its variants are also reported.The 327 aa mouse FAS polypeptide has the identifier number P25446 inUniProtKB database. FAS polypeptides known in other animals include, butare not limited to, Old World monkey (e.g., Q9BDN4in the UniProtKBdatabase), Rhesus monkey (e.g., Q9BDP2in the UniProtKB database), rat(e.g., Q63199in the UniProtKB database), or cow (e.g., P51867in theUniProtKB database).

Based on the sequence variation in the FAS polypeptide, a person ofordinary skill in the art can identify sequence variations in theeffector domain of the FAS polypeptide. For example, natural variants ofthe FAS effector domains can include one or more substitutions ormutations of C178R, L180F, P183L, I184V, T1981, Y232C, T241K, T241P,V249L, R250P, R250Q, G253D, G253S, N255D, A257D, I259R, D260G, D260V,D260Y, I262S, N264K, T270I, T270K, E272G, E272K, L278F, K299N, T3051,I310S, or any combinations thereof.

In one embodiment, an effector domain of the FAS polypeptide useful forthe invention comprises a death domain of the FAS polypeptide. Inanother embodiment, an effector domain of the FAS polypeptide comprises,consists essentially of, or consists of an amino acid sequence at least60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to aminoacids 230 to 314 of SEQ ID NO: 6. In other embodiments, an effectordomain of the FAS polypeptide comprises an intracellular domain of theFAS polypeptide. In yet other embodiments, an effector domain of the FASpolypeptide comprises an amino acid sequence at least 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 185 to335, amino acids 186 to 335, amino acids 187 to 335, amino acids 188 to335, amino acids 189 to 335, amino acids 190 to 335, amino acids 191 to335, amino acids 192 to 335, amino acids 193 to 335, amino acids 194 to335, amino acids 195 to 335, amino acids 196 to 335, amino acids 197 to335, amino acids 198 to 335, or amino acids 199 to 335 of SEQ ID NO: 6.

In still other embodiments, the effector domain of the FAS polypeptidefurther comprises a trans-membrane domain of the FAS polypeptide. In yetother embodiments, an effector domain of the FAS polypeptide comprisesan amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to amino acids 174 to 335 of SEQ ID NO: 6.In some embodiments, an effector domain of the FAS polypeptide furthercomprises about ten, about nine, about eight, about seven, about six,about five, about four, about three, about two, or about one amino acidfrom the C-terminal portion of the FAS extracellular domain. In certainembodiments, an effector domain of the FAS polypeptide comprises anamino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to amino acids 179 to 335, amino acids 178 to335, amino acids 177 to 335, amino acids 176 to 335, amino acids 175 to335, amino acids 174 to 335, amino acids 173 to 335, amino acids 172 to335, amino acids 171 to 335, amino acids 170 to 335, amino acids 169 to335, amino acids 168 to 335, amino acids 167 to 335, amino acids 166 to335, amino acids 165 to 335, amino acids 164 to 335, or amino acids 163to 335 of SEQ ID NO: 6, wherein the effector domain forms adeath-inducing signaling complex (DISC), activates caspase 8, or inducesapoptosis.

In some embodiments, an effector domain of the FAS polypeptidecomprises, consists essentially of, or consists of an amino acidsequence at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 8, wherein the effector domain forms adeath-inducing signaling complex (DISC), activates caspase 8, or inducesapoptosis.

In other embodiments, an effector domain of the FAS polypeptide isencoded by a nucleotide sequence at least 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

In one embodiment, the FAS-chimera gene product for the inventioncomprises, consists essentially of, or consists of an amino acidsequence at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 10, wherein the FAS-chimera gene product inducesapoptosis. In another embodiment, the FAS-chimera gene product isencoded by a nucleotide sequence at least 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to SEQ ID NO: 9, wherein theFAS-chimera gene product induces apoptosis.

B. Endothelial Cell-Specific Promoter

The nucleic acid construct comprising a FAS-chimera gene furthercomprises one or more expression control elements useful for regulatingthe expression of an operably linked FAS-chimera gene. The expressioncontrol elements include, but are not limited to, promoters, secretionsignals, and other regulatory elements.

The nucleic acid construct useful for the present invention utilizes anendothelial cell-specific promoter to direct expression of theFAS-chimera protein in an endothelial cell, thereby inducing apoptosisof the endothelial cell.

For the purpose of the present invention, an endothelial cell-specificpromoter can contain one or more cis-regulatory elements, which improvethe endothelial cell-specificity of the promoters compared to thepromoter without the cis-regulatory elements. In one example, thecis-regulatory element comprises an enhancer. In another aspect, thecis-regulatory element comprises a hypoxia response element. In otherexamples, the cis-regulatory element comprises both an enhancer and ahypoxia response element.

In one embodiment, a cis-regulatory element useful for the inventioncomprises a nucleotide sequence at least 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to SEQ ID NO: 11 or SEQ ID NO: 12 (thecomplementary sequence of SEQ ID NO: 11), wherein the cis-regulatoryelement improves endothelial cell specificity of a promoter compared toa promoter without the cis-regulatory element. The cis-regulatoryelement can further comprise an additional nucleotide sequence at least60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO: 13 or SEQ ID NO: 14 (the complementary sequence of SEQ ID NO: 13).

In another embodiment, a cis-regulatory element for the inventioncomprises a nucleotide sequence at least 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to SEQ ID NO: 13 or SEQ ID NO: 14 (thecomplementary sequence of SEQ ID NO: 13), wherein the cis-regulatoryelement improves endothelial cell specificity of a promoter compared toa promoter without the cis-regulatory element. The cis-regulatoryelement can further comprise an additional nucleotide sequence at least60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO: 11 or SEQ ID NO: 12 (the complementary sequence of SEQ ID NO: 11).

In other embodiments, a cis-regulatory element for the inventioncomprises a nucleotide sequence at least 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to SEQ ID NO: 15 or SEQ ID NO: 16 (thecomplementary sequence of SEQ ID NO: 15), wherein the cis-regulatoryelement improves endothelial cell specificity of a promoter compared toa promoter without the cis-regulatory element. In yet other embodiments,a cis-regulatory element for the nucleic acid construct comprises SEQ IDNO: 7 or any fragments, variants, derivatives, or analogs thereof,wherein the fragments, variants, derivatives, or analogs improveendothelial cell specificity of a promoter compared to a promoterwithout the cis-regulatory element.

In some embodiments, a cis-regulatory element for the inventioncomprises a nucleotide sequence at least 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to SEQ ID NO: 22 or SEQ ID NO: 23,wherein the cis-regulatory element improves endothelial cell specificityof a promoter compared to a promoter without the cis-regulatory element.In yet other embodiments, a cis-regulatory element for the nucleic acidconstruct comprises SEQ ID NO: 22 or SEQ ID NO: 23 or any fragments,variants, derivatives, or analogs thereof, wherein the fragments,variants, derivatives, or analogs improve endothelial cell specificityof a promoter compared to a promoter without the cis-regulatory element.

In other embodiments, a cis-regulatory element for the inventioncomprises a nucleotide sequence at least 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to SEQ ID NO: 24 or SEQ ID NO: 25,wherein the cis-regulatory element improves endothelial cell specificityof a promoter compared to a promoter without the cis-regulatory element.In yet other embodiments, a cis-regulatory element for the nucleic acidconstruct comprises SEQ ID NO: 24 or SEQ ID NO: 25 or any fragments,variants, derivatives, or analogs thereof, wherein the fragments,variants, derivatives, or analogs improve endothelial cell specificityof a promoter compared to a promoter without the cis-regulatory element.

Table 3 shows various cis-regulatory element sequences useful for theinvention.

TABLE 3 Endothelial Cell-Specific Cis-regulatory Elements and PromotersSEQ ID NOs Sequences SEQ IDctggagggtg actttgcttc tggagccagt acttcatact tttcatt NO: 11 SEQ IDaatgaaaagt atgaagtact ggctccagaa gcaaagtcac cctccag NO: 12 SEQ IDgtacttcata cttttcattc caatggggtg actttgcttc tgga NO: 13 SEQ IDtccagaagca aagtcacccc attggaatga aaagtatgaa gtac NO: 14 SEQ ID3X element NO: 15ctccagaagcaaagtcaccccattggaatgaaaagtatgaagtacaatgaaaagtatgaagtactggctccagaagcaaagtcaccctccagaagcaaagtcaccccattggaatgaaaagtat gaagtacSEQ ID 3x element (Complementary Sequence of SEQ ID NO: 15) NO: 16gtacttcatacttttcattccaatggggtgactttgcttctggagggtgactttgcttctggagccagtacttcatacttttcattgtacttcatacttttcattccaatggggtgactttgct tctggagSEQ ID PPE-1 Promoter NO: 17gtacgtgtacttctgatcggcgatactagggagataaggatgtgcctgacaaaaccacattgttgttgttatcattattatttagttttccttccttgctaactcctgacggaatctttctcacctcaaatgcgaagtactttagtttagaaaagacttggtggaaggggtggtggtggaaaagtagggtgatcttccaaactaatctggttccccgcccgccccagtagctgggattcaagagcgaagagtggggatcgtccccttgtttgatcagaaagacataaaaggaaaatcaagtgaacaatgatcagccccacctccaccccacccccctgcgcgcgcacaatacaatctatttaattgtacttcatacttttcattccaatggggtgactttgcttctggagaaactcttgattcttgaactctggggctggcagctagcaaaaggggaagcgggctgctgctctctgcaggttctgcagcggtctctgtctagtgggtgttttctttttcttagccctgcccctggattgtcagacggcgggcgtctgcctctgaagttagccgtgatttcctctagagccgggtcttatctctggctgcacgttgcctgtgggtgactaatcacacaataacattgtttagggctggaatgaagtcagagctgtttacccccactctataggggttcaatataaaaaggcggcggagaactgtccgagtcagaagcgttcctgcaccggcgctgagagcctgacccggtctgctccgctgtccttgcgcgctgcctcccggctgcccgcgacgctttcgccccagtggaagggccacttgctgcggccgc SEQ ID PPE-1-3X promoterNO: 18 gtacgtgtacttctgatcggcgatactagggagataaggatgtgcctgacaaaaccacattgttgttgttatcattattatttagttttccttccttgctaactcctgacggaatctttctcacctcaaatgcgaagtactttagtttagaaaagacttggtggaaggggtggtggtggaaaagtagggtgatcttccaaactaatctggttccccgcccgccccagtagctgggattcaagagcgaagagtggggatcgtccccttgtttgatcagaaagacataaaaggaaaatcaagtgaacaatgatcagccccacctccaccccacccccctgcgcgcgcacaatacaatctatttaattgtacttcatacttttcattccaatggggtgactttgcttctggagaaactcttgattcttgaactctggggctggcagctagcctccagaagcaaagtcaccccattggaatgaaaagtatgaagtacaatgaaaagtatgaagtactggctccagaagcaaagtcaccctccagaagcaaagtcaccccattggaatgaaaagtatgaagtacgctagcaaaaggggaagcgggctgctgctctctgcaggttctgcagcggtctctgtctagtgggtgttttctttttcttagccctgcccctggattgtcagacggcgggcgtctgcctctgaagttagccgtgatttcctctagagccgggtcttatctctggctgcacgttgcctgtgggtgactaatcacacaataacattgtttagggctggaatgaagtcagagctgtttacccccactctataggggttcaatataaaaaggcggcggagaactgtccgagtcagaagcgttcctgcaccggcgctgagagcctgacccggtctgctccgctgtccttgcgcgctgcctcccggctgcccgcgacgctttcgccccagtggaagggccacttgctgcggccgc SEQ IDggtgactttg cttctggag NO: 22 SEQ ID ctccagaagcaaagtcacc NO: 23 SEQ IDgtacttcata cttttcatt NO: 24 SEQ ID aatgaaaagtatgaagtac NO: 25 SEQ IDHypoxia Response element NO: 26 gcacgt

A cis-regulatory element for the present invention can be linked to apromoter upstream or downstream of the promoter or inserted between thetwo nucleotides in the promoter. The endothelial cell-specific promoterfor the present invention can utilize any promoters known in the art.For example, suitable promoters which can be utilized for the presentinvention include the endothelial-specific promoters: preproendothelin-1(PPE-1 promoter), US 2010/0282634, published Nov. 11, 2010; and WO2011/083464, published Jul. 14, 2011); the PPE-1-3× promoter (U.S. Pat.No. 7,579,327, U.S. Pat. No. 8,071,740, U.S. Pat. No. 8,039,261,052010/0282634, US 2007/0286845, WO 2011/083464, and WO2011/083466); theTIE-1 (S79347, S79346) and the TIE-2 (U53603) promoters [Sato T N, ProcNatl Acad Sci USA 1993 Oct. 15; 90(20):9355-8], the Endoglin promoter[Y11653; Rius C, Blood 1998 Dec. 15; 92(12):4677-90], the vonWillerbrand factor [AF152417; Collins C J Proc Natl Acad Sci USA 1987July; 84(13):4393-7], the KDR/flk-1 promoter [X89777, X89776; Ronicke V,Circ Res 1996 August; 79(2):277-85],The FLT-1 promoter [D64016 AJ224863;Morishita K, J Biol Chem 1995 Nov. 17; 270(46):27948-53], the Egr-1promoter [AJ245926; Sukhatme V P, Oncogene Res 1987 September-October;1(4):343-55], the E-selectin promoter [Y12462; Collins T J Biol Chem1991 Feb. 5; 266(4):2466-73], The endothelial adhesion moleculespromoters: ICAM-1 [X84737; Horley K J EMBO J 1989 October;8(10):2889-96], VCAM-1 [M92431; Iademarco M F, J Biol Chem 1992 Aug. 15;267(23): 16323-9], PECAM-1 [AJ313330 X96849; CD31, Newman P J, Science1990 Mar. 9; 247(4947): 1219-22], the vascular smooth-muscle-specificelements: CArG box X53154 and aortic carboxypeptidase-like protein(ACLP) promoter [AF332596; Layne M D, Circ Res. 2002; 90: 728-736] andAortic Preferentially Expressed Gene-1 [Yen-Hsu Chen J. Biol. Chem, Vol.276, Issue 50, 47658-47663, Dec. 14, 2001], all of which areincorporated herein by reference in their entireties.

In one embodiment, a promoter linked to the endothelial cell-specificelement comprises a nucleotide sequence at least 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99%, or 100% of SEQ ID NO: 17, wherein the promoterlinked to the element induces endothelial cell-specificity to the geneoperably linked to the promoter. In another embodiment, a promoterlinked to the endothelial cell-specific element comprises a fragment, avariant, a derivative, or an analog of a wild-type PPE-1 promoter,wherein said fragment, variant, derivative, or analog thereof inducesendothelial cell-specificity to the gene operably linked to thepromoter. In one example, the endothelial cell-specific element can beinserted between nucleotide residues 442 and 449 corresponding to SEQ IDNO: 17.

In further embodiments, an endothelial cell-specific promoter comprisesa hypoxia responsive element. A hypoxia responsive element (HRE) islocated on the antisense strand of the endothelin-1 promoter. Thiselement is a hypoxia-inducible factor-1 binding site that is requiredfor positive regulation of the endothelin-1 promoter (of the human, ratand murine gene) by hypoxia. Hypoxia is a potent signal, inducing theexpression of several genes including erythropoietin (Epo), VEGF, andvarious glycolytic enzymes. The core sequence (8 base pairs) isconserved in all genes that respond to hypoxic conditions and theflanking regions are different from other genes. The ET-I hypoxiaresponsive element is located between the GAT A-2 and the AP-1 bindingsites. In one example, a hypoxia response element comprises SEQ ID NO:26, a fragment, a variant, a derivative, or an analog thereof.

In other embodiments, an endothelial cell-specific promoter useful forthe invention comprises, consists essentially of, or consists of anucleotide sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,99%, or 100% of SEQ ID NO: 18, wherein the promoter linked to thecis-regulatory element induces endothelial cell-specificity to the geneoperably linked to the promoter. In another embodiment, an endothelialcell-specific promoter comprises a fragment, a variant, a derivative, oran analog of SEQ ID NO: 18, wherein said fragment, variant, derivative,or analog thereof induces endothelial cell-specificity to the geneoperably linked to the promoter.

Additional variations of the endothelial cell-specific promoters can befound at WO2011/083464, WO2011/083466, and WO2012/052423, which areincorporated herein by reference in their entireties.

The present invention also provides a novel promoter sequence comprisinga nucleotide sequence SEQ ID NO: 17. In one example, the promoterfurther comprises an endothelial cell-specific cis-regulatory element.In one example, the endothelian cell-specific cis-regulatory elementcomprises SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO: 25, SEQ ID NO: 26 or any fragments, derivatives,variants, or analogs thereof, wherein the fragments, derivatives,variants, or analogs thereof improve endothelial cell-specificity of thepromoter compared to a promoter without the cis-regulatory element. Inanother example, the promoter comprises a nucleotide sequence of SEQ IDNO: 18. The invention includes a nucleic acid construct comprising thenovel promoter and a heterologous nucleotide sequence. In oneembodiment, the heterologous nucleic acid sequence comprises anucleotide sequence encoding a FAS-chimera protein described herein. Inanother embodiment, the heterologous nucleotide sequence comprises anadenovirus sequence.

C. Vector

The invention also provides a vector comprising the nucleic acidconstruct, which comprises a FAS-chimera gene operably linked to anendothelial cell-specific promoter. For the purposes of this invention,numerous vector systems may be employed. For example, various viral genedelivery systems that can be used in the practice of this aspect of theinvention include, but are not limited to, an adenoviral vector, analphavirus vector, an enterovirus vector, a pestivirus vector, alentiviral vector, a baculoviral vector, a herpesvirus vector, anEpstein Barr viral vector, a papovaviral vector, a poxvirus vector, avaccinia viral vector, an adeno-associated viral vector and a herpessimplex viral vector.

In another embodiment, a vector comprising a FAS-chimera gene operablylinked to an endothelial cell-specific promoter is an adenovirus. Forexample, the adenovirus can be any one or more of human adenovirusspecies A (serotypes 12, 18, and 31), B (serotpyes 3, 7, 11, 14, 16, 21,34, 35, 50, and 55), C (serotypes 1, 2, 5, 6, and 57), D (8, 9, 10, 13,15, 17, 19, 20, 22-30, 32, 33, 36-39, 42-49, 51, 53, 54, and 56), E(serotype 4), F (serotype 40 and 41), or G (serotype 52). In aparticular embodiment, the adenovirus for the invention is humanadenovirus serotype 5. In some embodiments, the adenovirus useful forgene therapy is a recombinant non-replicating adenovirus, which does notcontain an E1 region and an E3 region. In certain embodiments, theadenovirus for the invention is a conditionally replicating adenovirus,which does not contain an E3 region, but contains an E1 region.

In one embodiment, the vector comprises, consists essentially of, orconsists of SEQ ID NO: 19. In another embodiment, the adenovirus vectoris an isolated virus having European Collection of Cell Cultures (ECACC)Accession Number 13021201.

D. Biological Deposits

Biological deposits were made with the European Collection of CellCultures (ECACC) located at Health Protection Agency CultureCollections, Health Protection Agency, Microbiology Services, PortonDown, Salisbury, SP4 0JG, UK, pursuant to the Budapest Treaty andpursuant to 37 C.F.R. §1.808. Samples of the deposited materials willbecome available to the public upon grant of a patent. The inventiondescribed and claimed herein is not to be limited by the scope of thestrain deposited, since the deposited embodiment is intended only as anillustration of the invention.

Strain ECACC Accession No. Date Deposited VB-111 13021201 Feb. 12, 2013

III. VEGF Antagonists

VEGF, vascular endothelial growth factor, is an endothelialcell-specific mitogen and an inducer of angiogenesis. The term VEGFencompasses the members of the VEGF gene family: VEGF-A, VEGF-B, VEGF-C,and VEGF-D. VEGF-A is considered the prototype member of the VEGF genefamily. Through alternative exon splicing, VEGF-A exists in fourdifferent isoforms: VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆. The fourVEGF-A isoforms are 121, 165, 189, and 206 amino acids in length(respectively) after signal sequence cleavage.

Once expressed, VEGF is secreted extracellularly where it binds to theextracellular region of a VEGF receptor (VEGFR). There are two primaryVEGFRs, VEGFR-1 or VEGFR-2, both of which are receptor tyrosine kinases.A third VEGFR, VEGFR-3, is a related receptor tyrosine kinase that onlybinds VEGF-C and VEGF-D. Upon binding to VEGF, the VEGFRs signaldownstream events that lead to endothelial cell proliferation andangiogenesis. VEGF-C and VEGF-D are known to regulate lymphaticangiogenesis.

The VEGF gene contains nucleotide sequences that are highly homologousto those of hypoxia-inducible factor-1 (HIF-1). These HIF-1 likesequences enable induction of VEGF gene expression under hypoxicconditions. Thus, under low oxygen conditions, such as within a tumormicroenvironment, VEGF gene expression is induced. The production ofhigh levels of VEGF within a tumor bed results in increased VEGFRsignaling and thus endothelial cell growth and angiogenesis. Theformation of new blood vessels within the tumor provides blood andoxygen to the growing tumor.

Due to the prominent role of VEGF in angiogenesis and tumor growth anddevelopment, VEGF antagonists are studied as potential cancertherapeutic agents. VEGF antagonists can prevent VEGF activity bybinding directly to VEGF and blocking its interaction with a VEGFR. Thisreduces signaling from the VEGFR and downstream events, thereby causinga reduction in angiogenesis. In one embodiment, a VEGF antagonist usefulfor the invention is an anti-VEGF antibody or a VEGF binding molecule.In another embodiment, an anti-VEGF antibody or VEGF-binding molecule isa monoclonal antibody, a humanized antibody, a human antibody, a singlechain antibody, or a chimeric antibody. In another embodiment, ananti-VEGF antibody or VEGF-binding molecule for the therapy comprisesFab, F(ab)₂, Fv, or scFv.

Another type of VEGF antagonist that can reduce or inhibit VEGF activityis a molecule binding to a VEGFR and thus blocking VEGFR interactionwith VEGF. This interference of receptor/ligand binding prevents VEGFRsignaling and reduces angiogenesis and endothelial cell proliferation.In one embodiment, the VEGF antagonist is an anti-VEGFR antibody orVEGFR-binding molecule. In another embodiment, the anti-VEGFR antibodyor VEGFR-binding molecule is a monoclonal antibody, a humanizedantibody, a human antibody, a single chain antibody, or a chimericantibody. In another embodiment, the anti-VEGFR antibody orVEGFR-binding molecule comprises Fab, F(ab)₂, Fv, or scFv.

VEGF antagonists that bind to VEGF or VEGFR can inhibit VEGF activity bysimilar mechanisms of action in that they prevent receptor/ligandinteraction, VEGFR signaling, and downstream signaling events such asendothelial cell proliferation and angiogenesis. Thus, in oneembodiment, the VEGF antagonist is selected from the group consisting ofbevacizumab (U.S. Pat. No. 7,169,901, incorporated herein by referencein its entirety), ranibizumab (U.S. Pat. No. 7,297,334, incorporatedherein by reference in its entirety), VGX-100 (U.S. Pat. No. 7,423,125,incorporated herein by reference in its entirety), r84 (U.S. Pat. No.8,034,905, incorporated herein by reference in its entirety),aflibercept (U.S. Pat. No. 5,952,199, incorporated herein by referencein its entirety), IMC-18F1 (U.S. Pat. No. 7,972,596, incorporated hereinby reference in its entirety), IMC-1C11 (PCT/US2000/02180, incorporatedherein by reference in its entirety), and ramucirumab (U.S. Pat. No.7,498,414, incorporated herein by reference in its entirety). A VEGFbinding molecule includes other forms of antibody derived molecules,e.g., a monobody, diabody, minibody, or any chimeric proteins comprisingat least one CDR of a VEGF binding antibody, e.g., bevacizumab.

In one embodiment, the anti-VEGF antibody or VEGF binding moleculecomprises at least one CDR selected from the group consisting of V_(H)CDR1 (SEQ ID NO: 28), V_(H) CDR2 (SEQ ID NO: 29), V_(H) CDR3 (SEQ ID NO:30), V_(L) CDR1 (SEQ ID NO: 31), V_(L) CDR2 (SEQ ID NO: 32), V_(L) CDR3(SEQ ID NO: 33), and any combination thereof. See Table 4.

TABLE 4 Amino Acid Sequences of Complementarity Determining Regions CDRSequence V_(H) CDR1 (SEQ ID NO: 28) GYTFTNYGMNV_(H) CDR2 (SEQ ID NO: 29) WINTYTGEPTYAADFKR V_(H) CDR3 (SEQ ID NO: 30)YPHYYGSSHWYFDV V_(L) CDR1 (SEQ ID NO: 31) SASQDISNYLNV_(L) CDR2 (SEQ ID NO: 32) FTSSLHS V_(L) CDR3 (SEQ ID NO: 33) QQYSTVPWT

In another embodiment, the anti-VEGF antibody or the VEGF bindingmolecule comprises CDR1 (SEQ ID NO: 28), CDR2 (SEQ ID NO: 29), or CDR3(SEQ ID NO: 30) of the heavy chain variable region (V_(H)) ofbevacizumab. For example, an anti-VEGF antibody or VEGF binding moleculecomprises CDR1 and CRD2 of V_(H), CDR 1 and CDR3 of V_(H), CDR2 and CDR3of V_(H), or CDR1, CDR2, or CDR3 of V_(H). In other embodiments, theanti-VEGF antibody or the VEGF binding molecule comprises CDR1 (SEQ IDNO: 31), CDR2 (SEQ ID NO: 32), or CDR3 (SEQ ID NO: 33) of the lightchain variable region (V_(L)) of bevacizumab. For example, an anti-VEGFantibody or VEGF-binding molecule comprises CDR1 and CDR2 of V_(L), CDR1and CDR3 of V_(L), CDR2 and CDR3 of V_(L), or CDR1, CDR2, and CDR3 ofV_(L). In some embodiments, an anti-VEGF antibody or VEGF bindingmolecule comprises V_(H) of bevacizumab. In certain embodiments, ananti-VEGF antibody or VEGF binding molecule comprises V_(L) ofbevacizumab.

In another aspect of the present invention, the anti-VEGF antibody orVEGF binding molecule comprises V_(H) CDR1 (SEQ ID NO: 28), V_(H) CDR2(SEQ ID NO: 29), V_(H) CDR3 (SEQ ID NO: 30), V_(L) CDR1 (SEQ ID NO: 31),V_(L) CDR2 (SEQ ID NO: 32), and V_(L) CDR3 (SEQ ID NO: 33).

IV. Treatment Methods Using Adenovirus Expressing FAS-Chimera Proteinand a VEGF Antagonist

One embodiment of the present invention provides methods of inducing orimproving responsiveness to a VEGF antagonist in a subject or a subjectpopulation in need thereof comprising administering a vector expressinga FAS chimera protein in combination with a VEGF antagonist.

In one aspect, the present invention includes a method of inhibiting orreducing angiogenesis to a subject or subject population in need ofcomprising (i) identifying a subject or subject population to be acandidate for improved responsiveness to a VEGF antagonist treatment and(ii) administering a vector encoding a Fas chimera protein and the VEGFantagonist to the subject or the subject population, wherein the subjector subject population has improved responsiveness to a VEGF antagonistafter the administration of the vector.

In another aspect, the present invention includes a method of inhibitingor reducing angiogenesis to a subject or a subject population in need ofinduced or improved responsiveness to a VEGF antagonist comprisingadministering a vector encoding a Fas chimera protein and the VEGFantagonist to the subject or the subject population, wherein the subjector the subject population has improved responsiveness to a VEGFantagonist after the administration of the vector.

In other aspects, identifying a subject or a subject population to be acandidate for improved responsiveness to a VEGF antagonist comprises (a)administering a VEGF antagonist alone, (b) measuring responsiveness tothe VEGF antagonist, (c) administering a vector encoding a Fas chimeraprotein, (d) administering the VEGF antagonist, (e) measuringresponsiveness to the VEGF antagonist in (d), and (f) comparing theresponsiveness in (b) with the responsiveness in (d). The subject or thesubject population demonstrating a higher responsiveness in (e) than (b)can be identified as a subject in need of induced or improvedresponsiveness to the VEGF antagonist. The subject or the subjectpopulation demonstrating an equal responsiveness between (b) and (e) ora higher responsiveness in (b) than (e) can be identified as anon-candidate and can be excluded from further therapy.

In one embodiment, the invention is directed to a method of inducingapoptosis of an endothelial cell in a tumor of a subject in need thereofcomprising administering a VEGF antagonist to the subject, wherein thesubject is administered with a vector which comprises a Fas-chimera geneoperably linked to an endothelial cell specific promoter prior to theadministration of the VEGF antagonist and wherein the responsiveness ofthe subject to the VEGF antagonist is increased after administration ofthe vector. In another embodiment, the invention provides a method ofinducing apoptosis of an endothelial cell in a tumor of a subject inneed thereof comprising: (i) administering a vector which comprises aFas-chimera gene operably linked to an endothelial cell specificpromoter, wherein the responsiveness of the subject to a VEGF antagonistis increased after administration of the vector, and (ii) administeringthe VEGF antagonist to the subject. In other embodiments, the inventionincludes a method of inducing apoptosis of an endothelial cell in atumor of a subject in need thereof comprising (i) administering a vectorwhich comprises a Fas-chimera gene operably linked to an endothelialcell specific promoter, (ii) administering a VEGF antagonist to thesubject, and (iii) measuring responsiveness of the subject to the VEGFantagonist, wherein the responsiveness of the subject is increased afterthe administration of the vector. In yet other embodiments, the tumorsize of the subject is reduced after the administration of the VEGFantagonist.

In certain embodiments, the invention provides a method of reducing thesize of a tumor in a subject in need thereof comprising administering aVEGF antagonist to the subject, wherein the subject is administered witha vector which comprises a Fas-chimera gene operably linked to anendothelial cell specific promoter prior to the administration of theVEGF antagonist, wherein the responsiveness of the subject to the VEGFantagonist is increased after administration of the vector and beforeadministration of the VEGF antagonist, and wherein the tumor size in thesubject is reduced after administration of the VEGF antagonist. In someembodiments, the invention provides a method of reducing the size of atumor in a subject in need thereof comprising: (i) administering avector which comprises a Fas-chimera gene operably linked to anendothelial cell specific promoter, wherein the responsiveness of thesubject to the VEGF antagonist is increased after the administration ofthe vector, and (ii) administering a VEGF antagonist to the subject,wherein the tumor size in the subject is reduced after administration ofthe VEGF antagonist. The present invention also includes a method ofreducing the size of a tumor in a subject in need thereof comprising:(i) administering a vector which comprises a Fas-chimera gene operablylinked to an endothelial cell specific promoter, (ii) administering aVEGF antagonist to the subject; and (iii) measuring the responsivenessof the subject to the VEGF antagonist, wherein the responsiveness of thesubject to the VEGF antagonist is increased after the administration ofthe vector. In other embodiments, the tumor size is reduced compared tothe tumor size of a subject who is not administered with the vectorprior to the administration of the VEGF antagonist. In yet otherembodiments, the tumor size is measured by comparing the size of thetumor prior to the administration of the VEGF antagonist and the size ofthe tumor after the administration of the VEGF antagonist.

In certain embodiments, the invention includes a method of treating adisease or condition associated with tumor in a subject comprisingadministering a VEGF antagonist to a subject in need thereof, whereinthe subject is administered with a vector which comprises a Fas-chimeragene operably linked to an endothelial cell specific promoter prior tothe administration of the VEGF antagonist and wherein the responsivenessof the subject to the VEGF antagonist is increased after administrationof the vector and before administration of the VEGF antagonist. Theinvention is also directed to a method of treating a disease orcondition associated with tumor in a subject comprising (i)administering a vector which comprises a Fas-chimera gene operablylinked to an endothelial cell specific promoter, wherein theresponsiveness of the subject to a VEGF antagonist is increased afterthe administration of the vector, and (ii) administering the VEGFantagonist to the subject. In some embodiments, the invention provides amethod of treating a disease or condition associated with tumor in asubject comprising (i) administering a vector which comprises aFas-chimera gene operably linked to an endothelial cell specificpromoter, (ii) administering a VEGF antagonist to the subject; and (iii)measuring responsiveness of the subject to the VEGF antagonist, whereinthe responsiveness of the subject to the VEGF antagonist is increasedafter administration of the vector. In other embodiments, the tumor ofthe subject is progressed after the administration of the vector.

In some embodiments, the invention includes a method of identifying acandidate for a VEGF antagonist therapy comprising (i) measuring a tumorsize of a subject who is diagnosed as having a tumor prior toadministering a vector which comprises a Fas-chimera gene operablylinked to an endothelial cell specific promoter to the subject and (ii)measuring progression of the tumor after the administration of thevector, wherein the subject is identified as a candidate after the tumoris progressed. In other embodiments, the invention provides a method ofidentifying a candidate for a VEGF antagonist therapy comprising (i)measuring a tumor size of a subject who is diagnosed as having a tumorprior to administering a vector which comprises a Fas-chimera geneoperably linked to an endothelial cell specific promoter to the subject,(ii) measuring progression of the tumor after the administration of thevector, wherein the subject is identified as a candidate after the tumoris progressed, and (iii) instructing a healthcare provider to administera VEGF antagonist to the subject. In certain aspects, the tumorprogression is measured by the growth of the tumor. In some embodiments,clinical progression is assessed by clinical deterioration in thepatients neurological signs and symptoms such as muscle weakness andmental status.

In a particular embodiment, the growth of the tumor is measured by MRI.In other embodiments, the tumor of the subject is a recurrent tumor thatarose during treatment with the vector. In yet other embodiments, thetumor of the subject is a metastatic tumor that arose during treatmentwith the vector.

The term “subject” or “individual” or “animal” or “patient” or “mammal,”is meant any subject, particularly a mammalian subject, having or beingexpected to have increased or improved responsiveness to a VEGFantagonist. In some embodiments, the subject is meant any subjectpreviously non-responsive to a VEGF antagonist, but becomes responsiveto the VEGF antagonist after exposure to the vector. In one embodiment,the subject is a human. In another embodiment, the subject is a cancerpatient.

The phrase “subject or subject population in need of induced or improvedresponsiveness to a VEGF antagonist” as used herein indicates that thesubject or the subject population has been identified as a candidate orcandidates for the combination therapy. The subject or subjectpopulation can be identified as being a candidate or candidates for thecombination therapy prior to the administration of the vector or theVEGF antagonist.

In certain embodiments, identifying a candidate for the combinationtherapy comprises measuring various characteristics of tumorangiogenesis, for example, reduction in size of the tumor, inhibition oftumor growth, reduction in angiogenesis, reduction inneo-vascularization, or any known characteristics of angiogenesis.

In one aspect, the subject or the subject population in need of improvedor induced responsiveness to a VEGF antagonist has a tumor or ametastasis thereof, wherein the VEGF antagonist and the vector treat,decrease, or reduce the size of a tumor or a metastasis thereof. Inanother aspect, the subject or the subject population in need ofimproved or induced responsiveness to a VEGF antagonist needsangiogenesis inhibition, wherein the vector and the VEGF antagonisttreat, decrease, prevent, or reduce angiogenesis. In other aspects, thesubject or the subject population in need of improved or inducedresponsiveness to a VEGF antagonist has cancer, wherein the vector andthe VEGF antagonist treat cancer.

In certain aspects, once the subject or the subject population isidentified as a candidate, the VEGF antagonist is administered prior toadministering the vector, concomitantly with administration of a vector,or after administration of a vector. In other aspects, after a subjector a subject population is identified as a candidate, the vector isadministered prior to the VEGF antagonist for at least one day earlier,at least two days earlier, at least three days earlier, at least fourdays earlier, at least five days earlier, at least six days earlier, atleast seven days earlier, at least nine days earlier, at least 10 daysearlier, at least two weeks earlier at least three weeks earlier, atleast four weeks earlier, at least one month earlier, at least twomonths earlier, or more.

In one embodiment of the present invention, the invention includes amethod of stabilizing a disease or disorder associated with cancer. Insome embodiments, the invention includes a method of stabilizing adisease or disorder associated with metastatic colorectal cancer (mCRC),advanced nonsquamous non-small cell lung cancer (NSCLC), metastaticrenal cell carcinoma (mRCC), glioblastoma multiforme (GBM), Mülleriancancer, ovarian cancer, peritoneal cancer, fallopian tube cancer, oruterine papillary serousspects, the present invention reduces the volumeof malignant peritoneal fluid, e.g., ascites, reduces pain to thesubject, prolongs survival of the subject, or any combinations thereof.The tumor that can be reduced, inhibited, or treated with thecombination of the vector and the VEGF antagonist can be a solid tumor,a primary tumor, or a metastatic tumor. The term “metastatic” or“metastasis” refers to tumor cells that are able to establish secondarytumor lesions in another parts or organ.

A “solid tumor” includes, but is not limited to, sarcoma, melanoma,carcinoma, or other solid tumor cancer. “Sarcoma” refers to a tumorwhich is made up of a substance like the embryonic connective tissue andis generally composed of closely packed cells embedded in a fibrillar orhomogeneous substance. Sarcomas include, but are not limited to,chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma,osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolarsoft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloromasarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma,endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma,fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma,Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma,immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma ofT-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma,angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parostealsarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma,synovial sarcoma, or telangiectaltic sarcoma.

The term “melanoma” refers to a tumor arising from the melanocyticsystem of the skin and other organs. Melanomas include, for example,acra-lentiginous melanoma, amelanotic melanoma, benign juvenilemelanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma,juvenile melanoma, lentigo maligna melanoma, malignant melanoma,metastatic melanoma, nodular melanoma, subungal melanoma, or superficialspreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas include, for example, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypemephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tuberous carcinoma, verrucous carcinoma, or carcinoma viflosum.

Additional cancers that may be inhibited or treated include, forexample, Leukemia, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiplemyeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid, urinarybladder cancer, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, papillary thyroid cancer, neuroblastoma, neuroendocrinecancer, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, cervical cancer, endometrial cancer, adrenal corticalcancer, prostate cancer, Müllerian cancer, ovarian cancer, peritonealcancer, fallopian tube cancer, or uterine papillary serous carcinoma.

In other embodiments, the subject has had up to three, up to two, or upto one previous line of chemotherapy. In yet other embodiments, thesubject has not had more than 3 prior lines of chemotherapy forrecurrent cancer.

In certain embodiments, the invention provides a method for inducing orimproving responsiveness to a vascular endothelial growth factor (VEGF)antagonist in a subject or a subject population in need thereof,comprising administering a vector which comprises a Fas-chimera geneoperably linked to an endothelial cell specific promoter, and the VEGFantagonist to the subject or the subject population, wherein theresponsiveness to the VEGF antagonist is induced or improved after theadministration of the vector compared to the responsiveness to the VEGFantagonist without the administration of the vector.

In certain embodiments, once the subject or the subject population isidentified as a candidate, the method further comprises administeringrepeated doses of the VEGF antagonist and the vector.

The dose of the vector administered as part of the present invention canbe measured in virus particles (VPs). In one embodiment, the dose of thevector in the combination therapy with bevacizumab is lower than thedose that is used for the therapy without bevacizumab (e.g., a therapyusing the vector alone). For example, an effective amount of the vectorin the combination therapy with bevacizumab includes, but is not limitedto equal to or less than about 1×10¹³, 9×10¹², 8×10¹², 7×10¹², 6×10¹²,5×10¹², 4×10¹², 3×10¹², 2×10¹², 1×10¹², 9×10¹¹, 8×10¹¹, 7×10¹¹, 6×10¹¹,5×10¹¹, 4×10¹¹, 3×10¹¹, 2×10¹¹, 1×10¹¹, 9×10¹⁰, 8×10¹⁰, 7×10¹⁰, 6×10¹⁰,5×10¹⁰, 4×10¹⁰, 3×10¹⁰, 2×10¹⁰, or 1×10¹⁰ virus particles.

In one embodiment, the vector is administered at an effective amount ofat least about 1×10¹¹ virus particles. In another embodiment, the vectoris administered at an effective amount of at least about 1×10¹² virusparticles. In another embodiment, the vector is administered at aneffective amount of at least about 1×10¹³ virus particles. In anotherembodiment, the vector is administered at an effective amount of atleast about 1×10¹⁴ virus particles. In other embodiments, the vector isadministered at an effective amount of at least about 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, or 5×10¹⁰ virus particles.

The dose of the VEGF antagonist (e.g., bevacizumab) can be measured inmg/kg body weight. In one aspect, the dose of bevacizumab in thecombination therapy with the vector is lower than the dose ofbevacizumab without the vector (e.g., a therapy using bevacizumabalone). Non-limiting examples of an effective amount of bevacizumabinclude equal to or less than about 15 mg/kg, 14 mg/kg, 13 mg/kg, 12mg/kg, 11 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg,4 mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg.

In a specific embodiment, the vector is administered at an effectiveamount of 3×10¹² to 1×10¹³ VPs and bevacizumab is administered at aneffective amount of 5 mg/kg to 15 mg/kg.

The present invention provides methods of inducing or improvingresponsiveness to VEGF antagonists comprising administering a vector anda VEGF antagonist. The regimen used for administering the vector and theVEGF antagonist comprises repeated administration of the vector and thebevacizumab. In one embodiment, the vector is repeatedly administeredevery day, once in about 2 days, once in about 3 days, once in about 4days, once in about 5 days, once in about 6 days, once in about 7 days,once in about 2 weeks, once in about 3 weeks, once in about 4 weeks,once in about 5 weeks, once in about 6 weeks, once in about 7 weeks,once in about 2 months, or once in about 6 months. In another embodimentthe bevacizumab is repeatedly administered once in about 7 days, once inabout 2 weeks, once in about 3 weeks, once in about 4 weeks, once inabout 2 months, once in about 3 months, once in about 4 months, once inabout 5 months, or once in about 6 months. In a particular embodiment,the vector is administered every 2 months and bevacizumab isadministered every 2 weeks.

V. Pharmaceutical Compositions

Also provided in the invention is a pharmaceutical compositioncomprising a vector expressing a FAS-chimera protein used in the methodsof the invention. The pharmaceutical composition can be formulated foradministration to mammals, including humans. The pharmaceuticalcompositions used in the methods of this invention comprisepharmaceutically acceptable carriers, including, e.g., ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. In one embodiment, the composition is formulated by addingsaline.

The compositions of the present invention may be administered by anysuitable method, e.g., parenterally (e.g., includes subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional and intracranialinjection or infusion techniques), intraventricularly, orally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. As described previously, the compositioncomprising a nucleic acid construct which comprises a FAS-chimera genein an endothelial cell and thereby induces apoptosis of the endothelialcell. Accordingly, the composition can inhibit, reduce, or decrease thesize of a tumor or a metastasis thereof by inhibitingneo-vascularization and/or angiogenesis of the tumor endothelial cells.Likewise, the VEGF antagonist used in combination with the nucleic acidconstruct inhibit neo-vascularization and/or angiogenesis through directinhibition of VEGF activity. Therefore, in one embodiment, thecombination therapy is delivered systemically or locally. For systemicor local delivery, the pharmaceutical formulation containing the nucleicacid construct, the adenovirus, or the homogeneous population of theadenovirus can utilize a mechanical device such as a needle, cannula orsurgical instruments.

Sterile injectable forms of the compositions used in the methods of thisinvention may be aqueous or oleaginous suspension. These suspensions maybe formulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterile,injectable preparation may also be a sterile, injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example as a suspension in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents which arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation.

Parenteral formulations may be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionsmay be administered at specific fixed or variable intervals, e.g., oncea day, or on an “as needed” basis.

Certain pharmaceutical compositions used in the methods of thisinvention may be orally administered in an acceptable dosage formincluding, e.g., capsules, tablets, aqueous suspensions or solutions.Certain pharmaceutical compositions also may be administered by nasalaerosol or inhalation. Such compositions may be prepared as solutions insaline, employing benzyl alcohol or other suitable preservatives,absorption promoters to enhance bioavailability, and/or otherconventional solubilizing or dispersing agents.

An effective amount of the chemotherapeutic agent is available in theart. In one aspect, for example, an effective amount of bevacizumab canbe at least about 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, or 5 mg/kg.

EXAMPLES Example 1 Construction and Cloning of the Viral Vector

The vector was constructed using a backbone containing most of thegenome of adenovirus type 5, as well as partial homology to an adaptorplasmid, which enables recombination.

The E1 early transcriptional unit was deleted from the backbone plasmid,and further modified by deleting the pWE25 and the Amp resistanceselection marker site.

The adaptor plasmid, containing sequences of the Ad5, CMV promoter, MCS,and SV40 polyA was modified to delete deleting the CMV promoter, and thePPE-1 promoter and Fas-c fragment were inserted by restrictiondigestion. The modified PPE-1 promoter (PPE-1-3×, SEQ ID NO: 18) and theFas-chimera transgene (Fas-c, SEQ ID NO: 9) were utilized forconstruction of the adenoviral vector. The PPE-1-(3×)-Fas-c element(2115 bp) was constructed from the PPE-1-(3×)-luc element. This elementcontains the 1.4kb of the murine preproendothelin PPE-1-(3×) promoter,the Luciferase gene, the SV40 polyA site and the first intron of themurine ET-1 gene, originated from the pEL8 plasmid (8848 bp) used byHarats et al (Harats D. et al., JCI, 1995). The PPE-3-Luc cassette wasextracted from the pEL8 plasmid using the BamHI restriction enzyme. TheLuciferase gene was substituted by the Fas-c gene [composed of the extracellular and intra membranal domains of the human TNF-R1 (Tumor NecrosisFactor Receptor 1, SEQ ID NO: 4) and of the Fas (p55) intracellulardomain (SEQ ID NO: 8) (Boldin et al, JBC, 1995)] to obtain thePPE-1-3×-Fas-c cassette.

PPE-1 (3×)-Fas-c Plasmid—The cassette was further introduced into thebackbone plasmid by restriction digestion, resulting with the PPE-1(3×)-Fas-c plasmid.

Adaptor-PPE-1(3×)-Fas-c Plasmid—The PPE-1-3×-Fas-c element was extractedfrom the first generation construct PPE-1-3×-Fas-c plasmid, and wasamplified with designated PCR primers introducing SnaBl and EcoRlrestriction sites at the 5′-and-3′-end respectively. These sites wereused to clone the PPE-Fas-c fragment into the adaptor plasmid digestedwith SnaBl and EcoRl, resulting in the adaptor-PPE-1-3×-Fas-c used fortransfection of the host cells (for example, PER.C6 cells).

Example 2

Administration of Bevacizumab to Patients Previously Treated withAd5-PPE-1-3×-Fas-c

OBJECTIVES: The objectives of this study are: (i) to evaluate thesafety, tolerability, and efficacy of single and multiple doses ofVB-111 (1×10¹², 3×10¹², 1×10¹³ viral particles [VP]) in patients withrecurrent glioblastoma multiforme (GBM); (ii) to evaluate thedistribution of VB-111 after single and multiple IV infusions and thelevel of antibodies to the adenovirus vector; and (iii) to evaluate thesafety, tolerability, and efficacy of combination treatment of multipledoses of VB-111 (3×10¹², or 1×10¹³ viral particles) together withbevacizumab in patients with recurrent GBM. An example of such atreatment regimen is shown in FIG. 1.

VB-111 and bevacizumab are both anti-angiogenic agents that target thetumor vasculature. However, they do so based on two distinct MOAs:Bevacizumab antagonizes VEGF while VB-111 directly disrupts theangiogenic vessels.

Patient Selection Inclusion Criteria

Subjects must have a histologically confirmed diagnosis of glioblastomamultiforme or gliosarcoma. Subjects with recurrent disease whosediagnostic pathology confirmed glioblastoma multiforme or gliosarcomawill not need re-biopsy;

Measurable disease by RANO criteria (see Screening, Day −21-0); see alsoTable 8 in Example 4;

Subjects ≧18 years of age;

Disease progression or recurrence following standard of care treatmentwith temozolomide and radiation;

An interval of at least 4 weeks between prior surgical resection andstudy enrollment;

An interval of at least 12 weeks between prior radiotherapy or at least4 weeks from prior chemotherapy, and enrollment in this protocol;

Recovered to Grade 1 or less from the toxic effects of any earlierintervention;

Karnofsky performance status of at least 60%;

Adequate renal, liver, and bone marrow function according to thefollowing criteria:

-   -   Absolute neutrophil count ≧1500 cells/ml    -   Platelets ≧125,000 cells/ml    -   Total bilirubin within upper limit of normal (ULN)    -   Aspartate aminotransferase (AST)≦2.5× institutional ULN    -   Creatinine less than or equal to the ULN or creatinine clearance        ≧50 mL/min for patients with creatinine levels above normal        limits. (creatinine clearance calculated by the Cockcroft-Gault        formula, see Appendix II).    -   PT, PTT (in seconds) prolonged beyond >20% of the upper limits        of normal.

Subjects must be treated with corticosteroids on day 0. Subjects alreadyadministering steroids must be on a stable dose for at least 1 weekprior to entry with no anticipation of a need to increase the steroiddose throughout the study;

Ability to understand and willingness to sign a written informed consentdocument;

Males and females of childbearing potential must utilize a standardcontraception method throughout the course of the trial.

Cohorts 1 and 2 Additional Eligibility Criteria:

Subjects without major mass effect of tumor (defined as <5 mm shift, noherniation).

Cohorts 3-4 Additional Eligibility Criteria for Further Dosing:

Subject received VB-111 at least 2 months ago, and remains withoutevidence of disease progression for at least 2 months after dosing.Note: Subjects that received the first dose more than 2 months prior toapproval of this multiple dose amendment, may be eligible to receive the2nd dose later than 2 months after the first dose, provided they remainwith stable disease. Subjects who progressed later than 2 months afterthe baseline dose may be eligible for the compassionate use protocolVB-111-122-CU. Subjects who progress earlier than 2 months post dosingwill discontinue the study.

Subjects who experienced a VB-111 drug related adverse event, and arescheduled for a repeat dose, the repeat dose will be delayed until theseverity of the event is no more than CTCAE Grade 1.

Subjects who experienced a prolongation of PTT and are scheduled for arepeat dose, the repeat dose will be delayed until the PTT hasnormalized returned to within 20% of the baseline value, whether anypositive LAC or APLA test has normalized or not. Subjects withclinically significant thrombotic or bleeding events related to aprolonged PTT should not receive additional doses of VB-111.

Exclusion Criteria

Prior anti-angiogenic therapy including VEGF sequestering agents (iebevacizumab, aflibercept, etc) or VEGFR inhibitors (cedirinib,pazopanib, sunitinib, sorafenib, etc);

Prior stereotactic radiotherapy;

Pregnant or breastfeeding subjects;

Concomitant medication that may interfere with study results; e.g.

immunosuppressive agents other than corticosteroids;

Active infection;

Evidence of CNS haemorrhage CTCAE grade 2 or above on baseline MRI;

Expected to have surgery during study period;

Subjects who suffered from an acute cardiac event within the last 12months;

Subjects with active vascular disease, either myocardial or peripheral(i.e. acute coronary syndrome, cerebral stroke, transient ischemicattack or arterial thrombosis or symptomatic peripheral vascular diseasewithin the past 3 months);

Subjects with known proliferative and/or vascular retinopathy;

Subjects with known liver disease (alcoholic, drug/toxin induced,genetic, or autoimmune);

Subjects with known active second malignancy;

Subjects testing positive to one of the following viruses: HIV, HBV andHCV;

Subjects that have undergone major surgery within the last 4 weeksbefore enrollment;

Subjects may not have received any other investigational agent within 4weeks before enrollment;

Uncontrolled intercurrent illness including, but not limited to ongoingor active infection, symptomatic congestive heart failure, unstableangina pectoris, cardiac arrhythmia, or psychiatric illness/socialsituations that would limit compliance with study requirements.

Bevacizumab-Specific Exclusion Criteria

Inadequately controlled hypertension (defined as systolic bloodpressure >150 mmHg and/or diastolic blood pressure >100 mmHg) within 28days of first study treatment;

Prior history of hypertensive crisis, hypertensive encephalopathy,reverse posterior leukoencephalopathy syndrome (RPLS);

Prior history of gastrointestinal perforation or abscess;

Clinically significant (i.e. active) cardiovascular disease, for examplecerebrovascular accidents ≦6 months prior to study enrollment,myocardial infarction ≦6 months prior to study enrollment, unstableangina, New York Heart Association (NYHA) Grade II or greater congestiveheart failure (CHF), or serious cardiac arrhythmia uncontrolled bymedication or potentially interfering with protocol treatment;

History or evidence upon physical/neurological examination of centralnervous system disease (e.g. seizures) unrelated to cancer orpotentially interfering with protocol treatment (unless adequatelycontrolled by medication);

Significant vascular disease (e.g., aortic aneurysm requiring surgicalrepair or recent arterial thrombosis) within 6 months prior to start ofstudy treatment. Any previous venous thromboembolism >NCI commontoxicity criteria adverse event (CTCAE) Grade 3,

History of pulmonary hemorrhage/hemoptysis ≧grade 2 (defined as ≧2.5 mLbright red blood per episode) within 1 month of first study treatment;

History or evidence of inherited bleeding diathesis or significantcoagulopathy at risk of bleeding (i.e. in the absence of therapeuticanticoagulation);

Current or recent (within 10 days of study enrollment) use of aspirin(>325 mg/day), clopidogrel (>75 mg/day) or equivalent. Prophylactic useof anticoagulants is allowed;

Surgical procedure (including open biopsy, surgical resection, woundrevision, or any other major surgery involving entry into a body cavity)or significant traumatic injury within 28 days prior to first studytreatment, or anticipation of need for major surgical procedure duringthe course of the study;

Minor surgical procedure, e.g. stereotactic biopsy, within 7 days offirst study treatment; placement of a vascular access device, within 2days of first study treatment;

History of intracranial abscess within 6 months prior to first studytreatment;

History of active gastrointestinal bleeding within 6 months prior tofirst study treatment;

Serious, non-healing wound, active ulcer, or untreated bone fracture;

Known hypersensitivity to any component of bevacizumab or any of thestudy drugs.

Proteinuria at screening as demonstrated by either:

-   -   Urine protein: creatinine (UPC) ratio ≧1.0 at screening OR    -   Urine dipstick for proteinuria ≧2+(patients discovered to have        ≧2+ proteinuria on dipstick urinalysis at baseline should        undergo a 24 hour urine collection and must demonstrate ≦1 g of        protein in 24 hours to be eligible)

Treatment Plan

This will be a prospective, open label, dose escalating, multi-center,Phase 1/2 study, measuring the safety, tolerability, distribution, andefficacy of single or multiple doses of intravenously administeredVB-111 in patients with recurrent GBM.

Dose Cohorts

VB-111 will be administered as a single intravenous infusion of 1×10¹²(Cohort 1) or 3×10¹² VP (Cohort 2), or multiple intravenous infusionsconsisting of an initial infusion of 3×10¹² followed by subsequentinfusions of 1×10¹³ (Cohort 3) or multiple intravenous infusions of1×10¹³ (Cohort 4).

Eligible, consenting patients will be enrolled into one of foursequential dosing cohorts as follows:

TABLE 5 Dosing Plan Cohort VB-111 (vp) Patients No. 1 1 × 10¹² SingleDose 3-6 2 3 × 10¹² Single Dose 3-6 3 3 × 10¹² Initial Dose + 1 × 10¹³Minimum of 3 Repeat Dose Maximum of 29 4 1 × 10¹³ Multiple Doses Maximumof 49 3-4 EXT 1 × 10¹³(Q2months) + 10 mg/kg Maximum of 29 bevacizumab(Q2weeks)

The study will be conducted according to the Simon's 2 step method. Upto 90 subjects are anticipated to enroll in this study, with up to 29subjects in Cohort 3, and up to 49 subjects in Cohort 4.

Step one will include the first 10 evaluable patients at the 1×10¹³ VPdose level or MTD.

For efficacy analysis, evaluable patients will be defined as those whoreceived at least one repeat dose of 1×10¹³ VPs or of MTD, or patientswho progressed sooner than 2 months after an initial dose of 1×10¹³ VPs.

A subject will be considered to have a response if s/he is either aliveand progression free at 6 months or has at least a partial tumorresponse according to Rano criteria within 6 months post dosing. If lessthan 2 responses are observed in the step 1 subjects, step 2 will behalted, otherwise, an additional 19 subjects will enroll in step 2.

Dose Escalation and Dose-Limiting Toxicity

Toxicities will be graded according to the NCI common toxicity criteriaVersion 4.0. Dose limiting toxicity (DLT) is any grade 3 or highertoxicity judged drug-related that does not respond to maximal medicaltherapy.

Cohorts 1 & 2:

Three subjects were enrolled sequentially to receive 1×10¹² (cohort 1)and observed for 28 days for DLT monitoring. No DLTs occurred in cohort1 and the study proceeded with cohort 2.

Three subjects were enrolled sequentially to receive 3×10¹² (cohort 2)and observed for 28 days for DLT. No DLTs were reported in cohort 2 andthe study proceeded to cohort 3.

Cohort 3:

Study subjects were initially enrolled into this cohort to receive asingle dose. Upon approval of Protocol Version 6.0, subjects remainingstable began to receive bi-monthly doses of VB-111 followingestablishment of safety with repeat doses at a rate of 1×10¹³ VPs.

Cohort 4:

Study subjects will be enrolled sequentially into the multiple dosecohort 4 and will receive VB-111 at 1×10¹³ VPs every 2 months.

Cohorts 3-4 EXT:

Upon disease progression, subjects will receive a combination of VB-111and bevacizumab. The first subjects receiving this combination treatmentwill be monitored for DLTs according to the following:

Stage 1: A minimum of 3 subjects will receive bi-monthly doses of VB-111(3×10¹² VPs) and bi-weekly bevacizumab (10 mg/Kg).Stage 2: A minimum of 3 subjects will receive bi-monthly doses of VB-111(1×10¹³ VPs) and bi-weekly bevacizumab (10 mg/Kg).

Refer to Monitoring for Dose Limiting Toxicities below for details onDLT monitoring in Cohorts 3-4.

Monitoring for Dose Limiting Toxicities:

Monitoring for DLTs was performed to establish safety at the beginningof each dose level escalation (Cohorts 1-4; 1×10¹²-1×10¹³ VPs). No DLTSwere observed in this study to date, therefore, the study has proceededas planned.

For Cohort 3-4 EXT:

Stage 1: Upon each local IRB approval of this protocol amendment (7.0),the first three subjects who progress after receiving at least one doseof 1×10¹³ VPs (initial or repeat) will receive a combination of areduced dose of VB-111 (3×10¹² VPs) and bevacizumab (10 mg/kg). Thesesubjects will be monitored for DLTs following this first combinationdose.

[Since a minimum of 2 months is required between doses of VB-111, if asubject progresses before 2 months, the subject will begin treatmentwith bevacizumab (10 mg/kg) every 2 weeks and at 2 months from the lastdose of VB-111, the subject will receives the 3×10¹² VPs dose ofVB-111.]

Among the first 3 subjects enrolled, if 1 DLT is observed within 4 weeksfollowing the initial combination dose, 3 additional subjects willreceive a combination dose, and safety will be reassessed.

If two out of six subjects who have received a combination dose ofVB-111 (3×10¹² VPs) and bevacizumab (10 mg/kg) experience a DLT, dosingin the Cohort 3-4 EXT shall stop and all progressed subjects will befollowed for long term follow up and survival.

If no DLTs are observed or only 1 DLT is observed in 6 patients, furtherdosing will escalate to Stage 2 levels: VB-111 (1×10¹³ VPs) andbevacizumab (10 mg/kg), see below.

Three subjects were enrolled sequentially and received 3×10¹² VPs andbevacizumab (10 mg/kg) and observed for 28 days for DLTs. No DLTS wereobserved in this study to date, therefore, the study proceeded asplanned to Stage 2 combination therapy: VB-111 (1×10¹³ VPs) andbevacizumab (10 mg/kg), see below.

Stage 2:

DLT monitoring will be performed for a minimum of 3 subjects for 4 weeksfrom the first combination dose of VB-111 (1×10¹³ VPs) and bevacizumab(10 mg/Kg).

Among the first 3 subjects enrolled at Stage 2 dosing, if 1 DLT isobserved within 4 weeks following the initial combination dose, 3additional subjects receive a combination dose, and safety will bereassessed.

If two out of six subjects who have received a combination dose ofVB-111 (1×10¹³ VPs) and bevacizumab (10 mg/kg) experience a DLT, allfuture doses in the Cohort 3-4 EXT shall be reduced to VB-111 at 3×10¹²VPs and bevacizumab (10 mg/kg).

If no DLTs are observed or only 1 DLT is observed in 6 patients, allfuture Cohort 3-4 EXT dosing will proceed as planned: VB-111 (1×10¹³VPs) and bevacizumab (10 mg/kg).

In order to allow sufficient time to monitor for DLTs, scheduling ofcombination doses will require prior approval from VBL.

Three subjects were enrolled sequentially and received 1×10¹³ VPs andbevacizumab (10 mg/kg) and observed for 28 days for DLTs. No DLTS wereobserved in this study to date, therefore, all subjects proceeded toreceive combined therapy of VB-111 (1×10¹³ VPs) and bevacizumab (10mg/kg).

Study Visits Screening, Day −21-0

Prospective patients will be screened within 3 weeks of enrollment. As apatient is screened, he/she will be given a Patient IdentificationNumber (PIN) by the site study coordinator, which will be recorded in apatient screening log, and will be assessed for eligibility according tothe protocol selection criteria. Patients who meet the eligibilitycriteria will be offered a choice of participation. The PIN consists ofthe center-number (e.g. 01) plus the patient number in sequential order(e.g. 001, 002, 003, etc.), so the full PIN for center 1 will be 01001,01002, etc.

Patients will be informed of the advantages, risks and constraints ofthe study and will be asked to sign an informed consent form.

Patients will then be evaluated by medical history, physical examinationand laboratory assessment including ECG. The screening procedures willalso include recording the patient's height, weight, date of birth, sexand race.

Medical History: A routine medical history and physical examination willbe carried out within 4 weeks prior to enrollment. Baseline laboratoryevaluations are to be conducted within 4 weeks prior to start ofprotocol therapy and at the baseline visit prior to initiation oftherapy. Medical history will focus on previous and/or known illnesses,including possible known infections, such as HBV, HIV, HCV, or otherinfections within the last month, cardiac disease, and liver disease.Medications taken on a regular basis, including those taken in the lastmonth will be recorded. Chemotherapy and radiotherapy and/or immunesuppressive treatments taken in the previous 12 weeks will be recorded,as well as surgeries done within this time period. Women are asked forpregnancy and breastfeeding status.

Karnofsky Performance Status (KPS) and ECOG should be performed atScreening.

Physical Examination:

Physical Examination will focus on tumor organs and tumor measurements,according to RANO criteria, as well as the following: Head and neck;Eyes; Lungs; Heart; Abdomen; Joints; Peripheral circulation; Skin; andNeurologic.

Physical Examination should include collection of height and weight (atscreening visit only).

ECG: A standard 12-lead electrocardiogram with rhythm strip should beperformed.

Laboratory Analyses: Blood: Blood will be collected at a fasting stateand analyzed for the following:

-   -   Hematology: complete blood count with INR, PT and activated PTT    -   Chemistry: electrolytes, creatinine and blood urea, bilirubin,        alkaline phosphatase, ALT and AST; calcium, total protein, and        albumin,    -   Virus infection tests: HIV, HBV and HCV    -   A serum pregnancy test will be conducted in female patients with        childbearing potential

Urine: Routine urinalysis will be collected and analyzed withmicroscopic examination on positives

Vital Signs: The vital signs (supine systolic and diastolic bloodpressure, peripheral heart rate, temperature, respiration rate) will berecorded at screening.

Tumor Measure: Contrast and non-contrast brain MRI imaging will be doneat screening and within 72 hours of the baseline visit to assess extentof cancer. If there are less than 2 weeks between screening and baselinevisits, only 1 MRI will be required.

Pregnancy: Eligible female subjects will be informed of specialrestrictions with regards to pregnancy and breast feeding throughout thestudy period. Both male and female subjects will commit to use onestandard contraceptive method on a regular basis throughout the studyperiod starting from screening. Both men and women should not attemptpregnancy and women should not be pregnant or breast-feeding whileparticipating in this study. If sexually active, both men and womenshould use an effective method of birth control from the screening visitand for up to one year after treatment. Barrier contraceptives (condomsor diaphragms) with spermicide, intrauterine devices, hormonalcontraceptives (Depo-Provera, Norplant), oral contraceptive pills, andcomplete abstinence are examples of effective methods.

Final Patient Selection: The final decision about the eligibility of thepatient to be enrolled is to be made after all screening evaluations areavailable. All inclusion and exclusion criteria MUST be met. The reasonfor all screen failures will be captured via the Screening Log.

Baseline (Study Drug Administration), Day 0

VB-111 will be administered as an outpatient. Once the subject is foundeligible, he/she will be appointed to arrive at the clinic within 3weeks from screening in a fasting state until 30 minutes following studydrug administration.

Prior to dosing (D0): On the day of admission to the site, each subjectwill be verified for eligibility according to inclusion/exclusioncriteria (where applicable) and then tested within 24 hours prior todosing for the following evaluations.

Laboratory Analyses (According to the Operation Manual):

Blood samples will be collected for the following evaluations:

-   -   Hematology: hemoglobin, complete blood count with differential,        INR, PT and activated PTT;    -   Chemistry: electrolytes, creatinine and blood urea nitrogen,        bilirubin, alkaline phosphatase, ALT and AST, calcium, total        protein and albumin;    -   Antibody, biodistribution (VB-111 adenovirus DNA levels) and        transgene expression determination.    -   Angiogenic Biomarker/Cytokine samples:        -   Levels of von Willebrand factor (vWF) and TNFα (optional).        -   In addition, blood samples for TNF, sTNFRI, sTNFRII, VEGF,            FGF, IL-6, IL-8, E-selectin, ICAM-1

Urine will be collected for the following evaluations:

-   -   Routine urine analysis    -   Cohorts 1-2 only: Biodistribution (VB-111 adenovirus DNA levels)

Vital Signs: Vital signs (supine, systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded 15 minutes prior to dosing.

Anti-Pyretic Treatment: To avoid fever following study drugadministration, all patients will receive 1000 mg of acetaminophenstarting 1-2 hours prior to dosing followed by 500 mg PRN 24 hours.

Corticosteroid Treatment: To reduce potential edema response during drugadministration, dexamethasone treatment will be administered:

Initial dose with VB-111: 10 mg will be administered 30 minutes prior todosing, followed by 4 mg×2/day for 14 days post dosing. Subsequent doseswith VB-111: 10 mg will be administered 30 minutes prior to dosing,followed by 4 mg×2/day for 3 days post dosing. Further corticosteroidtreatment will be administered at Investigator's discretion. If subjectsbegin the study already on steroids, all efforts should be made by theInvestigator not to change the steroid dose within 5 days of diseaseassessment, unless clinically warranted. A decision to continue thesteroids or to begin tapering after this period of time is at thediscretion of the Investigator.

Study Drug Administration: Drug preparation and Infusion will be doneaccording to the operation manual. Maximum time for drug in saline is1.5 hours at room temperature. The vials should be opened in abiological safety cabinet. VB-111 will be infused to the patient at therelevant dosage according to the patient's weight, according to theOperations Manual. For patients less than 50 Kg of weight, a dose of3×10¹² or 1×10¹³ VPs will be reduced by 30%. VB-111 should beadministered at 1 ml/minute or 3 ml/minute (1×10¹³ VPs only). A regularmeal will be allowed 0.5 hour after dosing. As previously planned, thefirst three subjects who received a repeat dose of 1×10¹³ VPs (followinga previous dose of 3×10¹² VPs from cohort 3 or an initial dose of 1×10¹³VPs in cohort 4) were monitored in an in-patient setting for an 8 hourperiod after their infusions. No infusion rate-related adverse eventswere reported.

Up to 6 Hours Following Drug Administration, Day 0: Laboratory Analyses:

Biodistribution: Blood sample will be collected for VB-111 adenovirusDNA level expression determinations at the following time points:

-   -   0 (Prior to dosing, see above)    -   At end of infusion    -   3±0.5 hours    -   6±0.5 hours

Cohorts 1-2 only: Urine samples will be collected for VB-111 adenovirusDNA levels determinations:

-   -   0 (prior to dosing, see above)    -   Between 0-3 hours    -   Between 3-6 hours

Angiogenic Biomarker/Cytokine Samples: Blood samples will be collectedat 6 hours post dose for:

-   -   Levels of von Willebrand factor (vWF) and TNFα (optional).    -   In addition, blood samples for TNF, sTNFRI, sTNFRII, VEGF, FGF,        IL-6, IL-8, E-selectin, ICAM-1

Vital Signs: The vital signs (systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded at 30 and 60 minutes after dosing and at 4 and 6 hours postdosing, and/or upon disappearance of any adverse event, whichever comesfirst.

Adverse Events: Full supportive measures will be employed for allpatients with an adverse event. All adverse events occurring followingdrug administration will be documented in the case report forms (CRFs),together with the intensity, the therapeutic measures applied, theoutcome and the relationship to the investigational drug. Relatedadverse events will be followed through resolution. Unrelated adverseevents will be followed through resolution or end of study.

Concomitant Medications: There is no restriction on concomitantmedication, besides the drugs listed in the exclusion criteria. However,VB-111 should not be mixed with other drugs. All concomitant medicationadministered during the study will be documented from baselinethroughout participation or Early Termination visit.

Other Laboratory Analyses: Laboratory samples drawn in response to aclinically significant event will be documented as unscheduledlaboratory evaluations. In the event of clinically relevant abnormallaboratory values, the tests will be followed-up until the values havereturned to within normal range and/or an adequate explanation of theabnormality is found. All such laboratory investigations will beperformed at the study site, except for distribution assessments, whichwill be sent to an Independent Central Laboratory. Should any of theseresults require confirmation, re-testing will be performed in the samehospital laboratory where possible. Laboratory accreditationcertificates and normal reference ranges must be provided for eachhospital laboratory.

Follow Up for Cohorts 1 & 2

On Days 4±1, 7±1, 14±2, 28±3, 56±3, 84±3, 112±3, 140±3, 168±7/Earlytermination, each patient will be required to return to the clinic in afasting state, for the following evaluations.

Vital signs: Vital signs (supine systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded.

Laboratory Analyses: Blood sample will be collected for the followingevaluations:

-   -   Antibody, biodistribution (VB-111adenovirus DNA levels) and        transgene expression determination.    -   Hematology: hemoglobin, complete blood count, INR, PT, and        activated PTT    -   Chemistry: electrolytes, creatinine and blood urea nitrogen,        bilirubin, alkaline phosphatase, ALT and AST, calcium, total        protein and albumin

Laboratory samples drawn in response to a clinically significant eventwill be documented as unscheduled laboratory evaluations.

Urine samples will be collected for:

-   -   Routine urinalysis    -   VB-111 adenovirus DNA levels determinations

ECG: ECG will be performed on Day 28 and Day 168/early withdrawal visit(if prior to Day 28).

Tumor Measurement: Post study surveillance will include MRI scans every2 months until 1 year post dosing, and then every 3 months until 2 yearspost dosing, or until progression. Additionally, Vital signs will berecording at these time points.

Post discontinuation follow-up period: Patients who progress and/ordiscontinue the study will be followed up by a telephone contact every 2months for survival.

Follow Up for Cohorts 3 and 4: Day 4 Following D0 Dose, Days 56 and 112(i.e. Days 4, 60 and 116)

As of the date of approval of this amendment (Amendment 11), the Day 4visit is no longer required for any subjects in this trial. Prior to theapproval of this amendment, the following clinic visit was performed(italics):

On the 4th day following dose at D0, D56 and D112, each patient will berequired to return to the clinic in a fasting state for the followingevaluations:

Vital signs: Vital signs (supine systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded.

Laboratory Analyses: Blood samples will be collected for the followingevaluations:

-   -   Hematology: hemoglobin, complete blood count with differential,        INR, PT and activated PTT: In the case of PTT prolongation above        ULN, please refer to Section 6.2 Dose Delay/Modification for        further instructions.    -   Chemistry: electrolytes, creatinine and blood urea nitrogen,        bilirubin, alkaline phosphatase, ALT and AST, calcium, total        protein and albumin;    -   Antibody, biodistribution (VB-111 adenovirus DNA levels) and        transgene expression determination.    -   Angiogenic Biomarker/Cytokine samples:        -   Levels of von Willebrand factor (vWF) and TNFα (optional).        -   In addition, blood samples for TNF, sTNFRI, sTNFRII, VEGF,            FGF, IL-6, IL-8, E-selectin, ICAM-1

Urine will be collected for the following evaluations:

-   -   Routine urinalysis

Adverse Events: At the follow up visit, the patient will be questionedabout possible adverse events which may have occurred from the day oflast visit. All adverse events will be documented in the case reportforms (CRFs), together with the intensity, the therapeutic measuresapplied, the outcome and the relationship to the investigational drug.Related adverse events will be followed through resolution. Unrelatedadverse events will be followed through resolution or end of study.

Concomitant Medications: At the follow up visit to the investigator, thepatient will be questioned about possible medications which may havebeen taken from the day of last visit. All concomitant medications willbe recorded with generic name, indication, dosage, units, frequency,start and stop dates.

Following the analysis of the first 6 subjects participating in Cohort 3and the first 6 subjects participating in Cohort 4, it will bedetermined if this visit (Day 4 following Days 56 and 112), can beeliminated for each Cohort.

A Safety follow up telephone contact should be performed 14 daysfollowing the second dose (Day 56) visit for the first 3-6 subjects ineach Cohorts 3 and 4 to ensure no DLTs have occurred.

Follow Up for Cohorts 3 and 4: Safety Telephone Contact Every OtherMonth in Between Dosing Visits

As of the date of approval of this amendment (Version 7.0), the Days 28and 84 visits to the clinic are no longer required for any subjects inthis trial. A telephone call should be made every other month in betweendosing visits (days 28, 84, 140, etc.) to the subject to inquire aboutAEs and changes in medication.

Prior to the approval of this amendment, the following clinic visit wasperformed:

One month following the first two infusions (i.e. at D28 and Day 84),each patient will be required to return to the clinic in a fastingstate, for the following evaluations:

Vital signs: Vital signs (supine systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded.

Laboratory Analyses: Blood sample will be collected for the followingevaluations:

-   -   Hematology: hemoglobin, complete blood count, INR, PT, and        activated PTT: In the case of PTT prolongation above ULN, please        refer to Section 6.2 Dose Delay/Modification for further        instructions.    -   Chemistry: electrolytes, creatinine and blood urea nitrogen,        bilirubin, alkaline phosphatase, ALT and AST, calcium, total        protein and albumin    -   Antibody, biodistribution (VB-111adenovirus DNA levels) and        transgene expression determination.    -   Angiogenic Biomarker/Cytokine samples:        -   Levels of von Willebrand factor (vWF) and TNFα (optional).        -   In addition, blood samples for TNF, sTNFRI, sTNFRII, VEGF,            FGF, IL-6, IL-8, E-selectin, ICAM-1

Laboratory samples drawn in response to a clinically significant eventwill be documented as unscheduled laboratory evaluations.

Urine sample will be collected for: Routine urinalysis

Tumor Measurement: Subjects will be assessed for response using contrastand non-contrast brain magnetic resonance imaging (MRI) with assessmentbased on the RANO criteria.

Adverse Events: At the follow up visit to the investigator, the patientwill be questioned about possible adverse events which may have occurredfrom the day of last visit. Full supportive measures will be employedfor all patients with an adverse event. All adverse events will bedocumented in the case report forms (CRFs), together with the intensity,the therapeutic measures applied, the outcome and the relationship tothe investigational drug. Related adverse events will be followedthrough resolution. Unrelated adverse events will be followed throughresolution or end of study.

Concomitant Medications: At the follow up visit to the investigator, thepatient will be questioned about possible medications which may havebeen taken from the day of last visit. All concomitant medications willbe recorded with generic name, indication, dosage, units, frequency,start and stop dates.

Further VB-111 Administration Cohorts 3 and 4 (Every 2 Months FollowingInitial Dose: Day 56, Day 112, Day 168, Month 8, etc.)

On the day of admission to the site, each subject will be re-verifiedfor eligibility according to inclusion/exclusion criteria (whereapplicable) and then tested within 24 hours prior to dosing. Subjectswithout evidence of progressive disease will be considered for furtherdosing.

This visit and VB-111 dosing will be repeated every 2 months untildisease progression.

The following assessments will be performed:

Tumor Measure: Subjects will be assessed for response using contrast andnon-contrast brain magnetic resonance imaging (MRI) with assessmentbased on the RANO criteria. MRI imaging will be done up to 72 hoursprior to dosing to assess extent of cancer.

Physical Exam: Physical Examination will focus on tumor organs and tumormeasurements, according to RANO criteria, as well as the following: Headand neck; Eyes; Lungs; Heart; Abdomen; Joints; Peripheral circulation;Skin; and Neurologic.

ECG (Day 56 Only): A standard 12-lead electrocardiogram with rhythmstrip should be performed.

Laboratory Analyses (according to the operation manual): Blood samplewill be collected for the following evaluations:

-   -   Hematology: hemoglobin, complete blood count with differential,        INR, PT and activated PTT: In the case of PTT prolongation above        ULN, please refer to Section 6.2 Dose Delay/Modification for        further instructions.    -   Chemistry: electrolytes, creatinine and blood urea nitrogen,        bilirubin, alkaline phosphatase, ALT and AST, calcium, total        protein and albumin;    -   Antibody, biodistribution (VB-111 adenovirus DNA levels) and        transgene expression determination.    -   Angiogenic Biomarker/Cytokine samples will be collected prior to        dosing and 6 hours post dosing only when the patient is observed        for a minimum of 6 hours post dose (at the Investigator's        discretion):        -   Levels of von Willebrand factor (vWF) and TNFα (optional).        -   In addition, blood samples for TNF, sTNFRI, sTNFRII, VEGF,            FGF, IL-6, IL-8, E-selectin, ICAM-1    -   Serum pregnancy test

Urine will be collected for the following evaluation: Routine urineanalysis

Vital Signs: Vital signs (supine, systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded 15 minutes prior to dosing.

Anti-Pyretic Treatment: To avoid fever following study drugadministration, all patients will receive 1000 mg of acetaminophen 1-2hours prior to dosing followed by 500 mg every 4 hours for 24 hours.

Corticosteroid Treatment: To reduce potential edema response during drugadministration, dexamethasone treatment will be administered: Subsequentdoses with VB-111: 10 mg will be administered 30 minutes prior todosing, followed by 4 mg×2/day for 3 days post dosing. Furthercorticosteroid treatment will be administered at Investigator'sdiscretion. If subjects begin the study already on steroids, all effortsshould be made by the Investigator not to change the steroid dose within5 days of disease assessment, unless clinically warranted. A decision tocontinue the steroids or to begin tapering after this period of time isat the discretion of the Investigator.

Study Drug Administration: Infusion will be done according to theoperation manual. Prior to infusion, the saline should be brought toroom temperature. The vials should be opened in a biological safetycabinet and injected into normal saline for infusion according to theoperation manual. The final solution for administration should beadministrated not more than 90 minutes after preparation. VB-111 will beinfused to the patient at the relevant dosage according to the patient'sweight, as detailed in the Operations Manual. For patients less than 50Kg of weight, a dose of 3×10¹² or 1×10¹³ VPs will be reduced by 30%. Theintravenous infusions of diluted VB-111 should be administered at 1ml/minute or 3 mL/minute (1×10¹³ VPs only). A regular meal will beallowed 0.5 hour after dosing.

Adverse Events: Full supportive measures will be employed for allpatients with an adverse event. All adverse events occurring followingdrug administration will be documented in the case report forms (CRFs),together with the intensity, the therapeutic measures applied, theoutcome and the relationship to the investigational drug. Relatedadverse events will be followed through resolution. Unrelated adverseevents will be followed through resolution or end of study.

Concomitant Medications: There is no restriction on concomitantmedication, besides the drugs listed in the exclusion criteria. However,VB-111 should not be mixed with other drugs. All concomitant medicationadministered during the study will be documented from baseline until theDay 168 or Early Termination visit.

Other Laboratory Analyses: Laboratory samples drawn in response to aclinically significant event will be documented as unscheduledlaboratory evaluations. In the event of clinically relevant abnormallaboratory values, the tests will be followed-up until the values havereturned to within normal range and/or an adequate explanation of theabnormality is found. All such laboratory investigations will beperformed at the study site, except for distribution assessments, whichwill be sent to an Independent Central Laboratory. Should any of theseresults require confirmation, re-testing will be performed in the samehospital laboratory where possible. Laboratory accreditationcertificates and normal reference ranges must be provided for eachhospital laboratory.

Up to 6 Hours Following Further Drug Administration:

As of the date of approval of this amendment (Amendment 11), the 6 hourobservation period is no longer required for any subjects in this trial.

Prior to the approval of this amendment, the following visit schedulewas followed (italics):

Laboratory Analyses: Biodistribution: Blood sample will be collected forVB-111 adenovirus DNA level expression determinations at the followingtime points:

-   -   0 (Prior to dosing, see above)    -   At end of infusion    -   3±0.5 hours    -   6±0.5 hours

Angiogenic Biomarker/Cytokine Samples: Blood samples will be collectedat 6 hours post dose for:

-   -   Levels of von Willebrand factor (vWF) and TNFα (optional).    -   In addition, blood samples for TNF, sTNFRI, sTNFRII, VEGF, FGF,        IL-6, IL-8, E-selectin, ICAM-1

Vital Signs: The vital signs (systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded at 30 and 60 minutes after dosing and at 4 and 6 hours postdosing, and/or upon disappearance of any adverse event, whichever comesfirst.

Adverse Events: Full supportive measures will be employed for allpatients with an adverse event. All adverse events occurring followingdrug administration will be documented in the case report forms (CRFs),together with the intensity, the therapeutic measures applied, theoutcome and the relationship to the investigational drug. Relatedadverse events will be followed through resolution. Unrelated adverseevents will be followed through resolution or end of study.

Concomitant Medications: There is no restriction on concomitantmedication, besides the drugs listed in the exclusion criteria. However,VB-111 should not be mixed with other drugs. All concomitant medicationadministered during the study will be documented from baseline until theDay 168 or Early Termination visit.

Other Laboratory Analyses: Laboratory samples drawn in response to aclinically significant event will be documented as unscheduledlaboratory evaluations. In the event of clinically relevant abnormallaboratory values, the tests will be followed-up until the values havereturned to within normal range and/or an adequate explanation of theabnormality is found. All such laboratory investigations will beperformed at the study site, except for distribution assessments, whichwill be sent to an Independent Central Laboratory. Should any of theseresults require confirmation, re-testing will be performed in the samehospital laboratory where possible. Laboratory accreditationcertificates and normal reference ranges must be provided for eachhospital laboratory.

Upon Disease Progression: Cohort 3-4 Extension VB-111+BevacizumabAdministration Cohorts 3 and 4 (Every 2 Months Following DiseaseProgression)

At the time the subject experiences disease progression (any remainingstable subjects from cohort 3 and all subjects from cohort 4), thesubject will be asked to participate in an extension phase of thistrial, which will administer VB-111 and bevacizumab as a combinationtherapy. VB-111 (3×10¹² or 1×10¹³ VPs—see Dose Limiting Toxicitiesabove) will be administered bi-monthly and bevacizumab will beadministered bi-weekly.

Subjects will be informed of the advantages, risks and constraints ofthe study and will be asked to sign an informed consent form. Thensubjects will be evaluated to ensure they meet the study criteria. Referto Section 3.0 for Inclusion and Exclusion criteria, includingbevacizumab specific exclusion criteria.

Since a minimum of 2 months is required between doses of VB-111, if asubject progresses before 2 months, the subject will begin treatmentwith bevacizumab (10 mg/kg) every 2 weeks and at 2 months from the lastdose of VB-111, the subject will receive the first combination dose.]

The following assessments will be performed:

Tumor Measure; Subjects will be assessed for response using contrast andnon-contrast brain magnetic resonance imaging (MRI) with assessmentbased on the RANO criteria. MRI imaging will be done up to 72 hoursprior to dosing to assess extent of cancer.

Physical Exam: Physical Examination will focus on tumor organs and tumormeasurements, according to RANO criteria, as well as the following: Headand neck; Eyes; Lungs; Heart; Abdomen; Joints; Peripheral circulation;Skin; and Neurologic.

ECG (to be performed every 6 months from the initial combination dose.

A standard 12-lead electrocardiogram with rhythm strip should beperformed.

Laboratory Analyses (according to the operation manual): Blood samplewill be collected for the following evaluations:

-   -   Hematology: hemoglobin, complete blood count with differential,        INR, PT and activated PTT: In the case of PTT prolongation above        ULN, please refer to Section 6.2 Dose Delay/Modification for        further instructions.    -   Chemistry: electrolytes, creatinine and blood urea nitrogen,        bilirubin, alkaline phosphatase, ALT and AST, calcium, total        protein and albumin;    -   Antibody, biodistribution (VB-111 adenovirus DNA levels) and        transgene expression determination.    -   Angiogenic Biomarker/Cytokine samples are collected prior to        dosing and 6 hours post dosing only when the patient is observed        for a minimum of 6 hours post dose (at the Investigator's        discretion):        -   Levels of von Willebrand factor (vWF) and TNFα (optional).        -   In addition, blood samples for TNF, sTNFRI, sTNFRII, VEGF,            FGF, IL-6, IL-8, E-selectin, ICAM-1    -   Serum pregnancy test

Urine will be collected for the following evaluation:

-   -   Routine urine analysis    -   Dipstick for proteinuria

Vital Signs: Vital signs (supine, systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded 15 minutes prior to dosing.

Anti-Pyretic Treatment: To avoid fever following study drugadministration, all patients will receive 1000 mg of acetaminophen 1-2hours prior to dosing followed by 500 mg every 4 hours for 24 hours.

Corticosteroid Treatment: To reduce potential edema response during drugadministration, dexamethasone treatment will be administered: Subsequentdoses with VB-111: 10 mg will be administered 30 minutes prior todosing, followed by 4 mg×2/day for 3 days post dosing. Furthercorticosteroid treatment will be administered at Investigator'sdiscretion. If subjects begin the study already on steroids, all effortsshould be made by the Investigator not to change the steroid dose within5 days of disease assessment, unless clinically warranted. A decision tocontinue the steroids or to begin tapering after this period of time isat the discretion of the Investigator.

Bevacizumab Administration:

Bevacizumab will be administered by infusion at a dose of 10 mg/kgbefore VB-111 on dosing days. The rate of infusion shall be according tothe package insert for bevacizumab: The initial bevacizumab dose shouldbe delivered over 90 minutes as an IV infusion following chemotherapy.If the first infusion is tolerated, the second infusion may beadministered over 60 minutes. If the 60-minute infusion is tolerated,all subsequent infusions may be administered over 30 minutes.

Study Drug Administration:

Infusion will be done according to the operation manual. Prior toinfusion, the saline should be brought to room temperature. The vialsshould be opened in a biological safety cabinet and injected into normalsaline for infusion according to the operation manual. The finalsolution for administration should be administrated not more than 90minutes after preparation. VB-111 will be infused to the patient at therelevant dosage according to the patient's weight, as detailed in theOperations Manual. For patients less than 50 Kg of weight, a dose of3×10¹² or 1×10¹³ VPs will be reduced by 30%. The intravenous infusionsof diluted VB-111 should be administered at 1 ml/minute or 3 mL/minute(1×10¹³ VPs only).

Up to 8 Hours Following the Initial Combination of VB-111 andBevacizumab:

Laboratory Analyses: Biodistribution: Blood sample will be collected forVB-111 adenovirus DNA level expression determinations at the followingtime points:

-   -   0 (Prior to dosing, see above)    -   At end of infusion    -   3±0.5 hours    -   6±0.5 hours

Angiogenic Biomarker/Cytokine Samples: Blood samples will be collectedat 6 hours post dose for:

-   -   Levels of von Willebrand factor (vWF) and TNFα (optional).    -   In addition, blood samples for TNF, sTNFRI, sTNFRII, VEGF, FGF,        IL-6, IL-8, E-selectin, ICAM-1

Vital Signs: The vital signs (systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded at 30 and 60 minutes after dosing and at 4 and 6 hours postdosing, and/or upon disappearance of any adverse event, whichever comesfirst.

Adverse Events: Full supportive measures will be employed for allpatients with an adverse event. All adverse events occurring followingdrug administration will be documented in the case report forms (CRFs),together with the intensity, the therapeutic measures applied, theoutcome and the relationship to the investigational drug. Relatedadverse events will be followed through resolution. Unrelated adverseevents will be followed through resolution or end of study.

Concomitant Medications: There is no restriction on concomitantmedication, besides the drugs listed in the exclusion criteria. However,VB-111 should not be mixed with other drugs. All concomitant medicationadministered during the study will be documented from baselinethroughout participation or Early Termination visit.

Other Laboratory Analyses: Laboratory samples drawn in response to aclinically significant event will be documented as unscheduledlaboratory evaluations. In the event of clinically relevant abnormallaboratory values, the tests will be followed-up until the values havereturned to within normal range and/or an adequate explanation of theabnormality is found. All such laboratory investigations will beperformed at the study site, except for distribution assessments, whichwill be sent to an Independent Central Laboratory. Should any of theseresults require confirmation, re-testing will be performed in the samehospital laboratory where possible. Laboratory accreditationcertificates and normal reference ranges must be provided for eachhospital laboratory.

Follow Up Cohort 3-4 Extension Phase:

Bi-Weekly Treatment with Bevacizumab

Bevacizumab will be administered according to standard of care practicesbi-weekly. During the visits to the clinic to receive this treatment,the subject should be seen by the Investigator to assess safety.

Bevacizumab Administration

Bevacizumab will be administered by infusion at a dose of 10 mg/kgaccording to standard of care practices.

Adverse Events: Full supportive measures will be employed for allpatients with an adverse event. All adverse events occurring followingdrug administration will be documented in the case report forms (CRFs),together with the intensity, the therapeutic measures applied, theoutcome and the relationship to the investigational drug. Relatedadverse events will be followed through resolution. Unrelated adverseevents will be followed through resolution or end of study.

Concomitant Medications: There is no restriction on concomitantmedication, besides the drugs listed in the exclusion criteria. However,VB-111 should not be mixed with other drugs. All concomitant medicationadministered during the study will be documented from baselinethroughout participation or Early Termination visit.

Other Laboratory Analyses: Laboratory samples drawn in response to aclinically significant event will be documented as unscheduledlaboratory evaluations. In the event of clinically relevant abnormallaboratory values, the tests will be followed-up until the values havereturned to within normal range and/or an adequate explanation of theabnormality is found. All such laboratory investigations will beperformed at the study site, except for distribution assessments, whichwill be sent to an Independent Central Laboratory. Should any of theseresults require confirmation, re-testing will be performed in the samehospital laboratory where possible. Laboratory accreditationcertificates and normal reference ranges must be provided for eachhospital laboratory.

28 Days Following Initial Combination Treatment

One month following the initial combination treatment, each patient willbe required to return to the clinic in a fasting state, for thefollowing evaluations:

Vital signs: Vital signs (supine systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded.

Laboratory Analyses: Blood sample will be collected for the followingevaluations:

-   -   Hematology: hemoglobin, complete blood count, INR, PT, and        activated PTT: In the case of PTT prolongation above ULN, please        refer to Section 6.2 Dose Delay/Modification for further        instructions.    -   Chemistry: electrolytes, creatinine and blood urea nitrogen,        bilirubin, alkaline phosphatase, ALT and AST, calcium, total        protein and albumin    -   Laboratory samples drawn in response to a clinically significant        event will be documented as unscheduled laboratory evaluations.    -   Urine sample will be collected for:    -   Routine urinalysis    -   Dipstick for Proteinuria    -   No dose modifications for grade 1/2 events    -   Grade 3-(UPC>3.5, urine collection >3.5 g/24 hr, or dipstick        4+): Hold bevacizumab treatment until ≦Grade 2, as determined by        either UPC ratio ≦3.5 or 24 hr collection ≦3.5 g    -   Grade 4 (nephrotic syndrome): Discontinue bevacizumab

Adverse Events: At the follow up visit to the investigator, the patientwill be questioned about possible adverse events which may have occurredfrom the day of last visit. Full supportive measures will be employedfor all patients with an adverse event. All adverse events will bedocumented in the case report forms (CRFs), together with the intensity,the therapeutic measures applied, the outcome and the relationship tothe investigational drug. Related adverse events will be followedthrough resolution. Unrelated adverse events will be followed throughresolution or end of study.

Concomitant Medications: At the follow up visit to the investigator, thepatient will be questioned about possible medications which may havebeen taken from the day of last visit. All concomitant medications willbe recorded with generic name, indication, dosage, units, frequency,start and stop dates.

Study Completion/Early Termination

Subjects in Cohorts 3 and 4 will continue to receive VB-111 every 2months, provided that they continue to be stable. Once subjectsexperience disease progression, they may receive combination treatmentwith VB-111 or they will return to the clinic for a final study visit.In addition, if subjects withdraw consent or it is determined by theInvestigator that the subject should not continue in the study, thesubject will return for an Early Termination Visit. Subjects willcontinue to be followed every 2-3 months as part of their Standard ofCare follow up. VBL may request data on these subjects (e.g. MRI scans,other anti-cancer treatments).

Subjects who are no longer seen for standard of care visits at the siteshould be contacted by telephone every 2-3 months to follow up onsurvival and for information regarding further treatment forglioblastoma. Follow up will continue until the patient expires.

Duration of Study

Cohorts 1-2: Each patient will be administered with a single injectionof VB-111 and will be followed with regularly scheduled visits andevaluations for a period of 6 months after dosing or until progressionor study withdrawal and then followed for survival by a post studyfollow-up telephone contact every 2 months. Additionally, surveillanceMRIs will be performed every 2 months until 1 year post dosing, and thenevery 3 months until 2 years post dosing, Patients can decide towithdraw from study at any time. Patients who withdraw prior to day168±7 should be requested to return to the clinic for an earlytermination visit and should still be contacted and questioned aboutAEs. (Patients who withdraw after day 168 will not be considered earlyterminations.) All AEs, irrespective of whether related or not to thedisease, will be documented in the patient's record and CRF.

Cohorts 3 and 4: Subjects will be screened 3 weeks before the initialdose (D0) and will receive VB-111 every 2 months through diseaseprogression. Following disease progression, routine Standard of Carewill be performed; patients will be followed for disease status updatesand survival every 2-3 months.

Exploratory Assessments

Optional Biopsy Samples; Biopsies of brain tissue (preferably freshfrozen samples) may be collected for further testing by VBL as part of aclinically indicated procedure at any time in the study. If a sample ofbrain tissue is collected, a blood sample for transgene analysis shouldbe collected on the same day.

Circulating Endothelial Cells (CECs); Optionally, CECs may be collectedand analyzed as a biomarker for angiogenesis via Flow Cytometry at anytime in the study, if the subject consents and the site has localcapability to perform such testing.

Additional Exploratory Testing: Samples collected for plasma, serum,tissue throughout the study will be stored by VBL or its designee for upto 15 years for exploratory testing to better understand the impact,potential response and toxicity of VB-111.

Expected Adverse Events/Dose Modifications Expected Adverse EventsPre-Clinical Studies

VB-111 caused minimal toxicity in preclinical toxicology studies inmice. Mild anemia, mild thrombocytopenia, mild leukocytosis,splenomegaly, and bone marrow hyperplasia were observed. Transient liverenzyme elevations with no correlation with clinical pathology were alsoobserved.

Adenovirus Vectors and Anti-angiogenic Agents

The administration of adenovirus vectors systemically has been welltolerated.

Flu-like symptoms (fever, fatigue, rigors, nausea, and/or vomiting) arethe most common adverse events. The majority of intravenously injectedadenovirus particles are sequestered by the liver, which in turn causesan inflammatory response characterized by acute transaminitis andvascular damage.

The administration of anti-angiogenic agents systemically has also beenwell tolerated. The major predicted adverse effects have been woundhealing disorders, bleeding and thromboembolic events. Hypertension andproteinuria have also been reported with antiangiogenic therapy.

Expected Adverse Events with Bevacizumab

Gastrointestinal (GI) perforation

Serious and sometimes fatal GI perforation occurs at a higher incidencein bevacizumab-treated patients compared to controls

The incidences of GI perforation ranged from 0.3% to 2.4% acrossclinical studies

Discontinue bevacizumab in patients with GI perforation

Surgery and Wound Healing Complications

The incidence of wound healing and surgical complications, includingserious and fatal complications, is increased in bevacizumab-treatedpatients

Do not initiate bevacizumab for at least 28 days after surgery and untilthe surgical wound is fully healed. The appropriate interval betweentermination of bevacizumab and subsequent elective surgery required toreduce the risks of impaired wound healing/wound dehiscence has not beendetermined

Discontinue bevacizumab at least 28 days prior to elective surgery andin patients with wound healing complications requiring medicalintervention

Hemorrhage

Severe or fatal hemorrhage, including hemoptysis, GI bleeding,hematemesis, central nervous system hemorrhage, epistaxis, and vaginalbleeding, occurred up to 5-fold more frequently in patients receivingbevacizumab. Across indications, the incidence of grade ≧3 hemorrhagicevents among patients receiving bevacizumab ranged from 1.2% to 4.6%

Do not administer bevacizumab to patients with serious hemorrhage orrecent hemoptysis (≧½ tsp of red blood)

Discontinue bevacizumab in patients with serious hemorrhage (ie,requiring medical intervention)

Additional Serious Adverse Events

Additional serious and sometimes fatal adverse events with increasedincidence in the bevacizumab-treated arm vs control included

Non-GI fistula formation (≦0.3%)

Arterial thromboembolic events (grade ≧3, 2.6%)

Proteinuria (nephrotic syndrome, <1%)

Additional serious adverse events with increased incidence in thebevacizumab-treated arm vs control included

Hypertension (grade 3-4, 5%-18%)

Reversible posterior leukoencephalopathy syndrome (RPLS) (≦0.1%)

Infusion reactions with the first dose of bevacizumab were uncommon(≦3%), and severe reactions occurred in 0.2% of patients

Inform females of reproductive potential of the risk of ovarian failureprior to starting treatment with bevacizumab

Most common adverse reactions observed in bevacizumab patients at arate >10% and at least twice the control arm rate were epistaxis,headache, hypertension, rhinitis, proteinuria, taste alteration, dryskin and rectal hemorrhage.

Dosing Delays/Dose Modification

For subjects participating in multiple dose cohorts, dosing may bedelayed in the following situations:

-   -   Subjects who experienced a VB-111 drug related adverse event,        and are scheduled for a repeat dose, the repeat dose will be        delayed until the severity of the event is no more than CTCAE        Grade 1.    -   Subjects who experienced a prolongation of aPTT and are        scheduled for a repeat dose, the repeat dose will be delayed        until the PTT has returned to within 20% of the baseline value,        whether any positive LAC or APLA test has normalized or not.        Borderline cases may be discussed with the sponsor on a case by        case basis. Subjects with clinically significant thrombotic or        bleeding events related to a prolonged PTT should not receive        additional doses of VB-111.        -   In case of PTT prolongation above ULN, blood should be drawn            for lupus anticoagulant (LAC) and for anti-phospholipid            antibody (IgG and IgM for beta-2-GP-1 and anticardiolipin).            Patients with prolonged aPTT should not receive VB-111 until            the abnormal laboratory values return to within 20% of the            baseline value, whether any positive LAC or APLA test has            normalized or not. If repeat test for LAC/APLA remains            positive, it must be repeated every 12 weeks (from the            initial positive test) until it returns to normal.    -   During DLT monitoring. To comply with protocol restrictions        regarding 14 day observation periods between patients

Patients will be assigned to Cohort 3 (single or multiple doses of3×10¹² VPs) or

Cohort 4 (multiple doses of 1×10¹³ VPs). No dose modification will bepermitted for this trial. However, VB-111 will be infused to the patientat the relevant dosage according to the patient's weight, as detailed inthe Operations Manual. For patients less than 50 Kg of weight, a dose of3×10¹² or 1×10¹³ VPs will be reduced by 30%. The intravenous infusionsof diluted VB-111 should be administered at 1 ml/minute or 3 mL/minute(1×10¹³ VPs only).

Correlative/Special Studies

Distribution: For distribution assessment, blood samples will becollected from all patients according to the operation manual. Testingof these samples for Adenovirus and VB-111 transgene level determinationwill be conducted at the maximal tolerated dose group or the highestdose cohort. Distribution will be assessed by determination of levels ofviral DNA and transgene by Q-PCR and Q-RT-PCR respectively in the blood,at pre-determined time points following dosing. Samples will becollected for all patients at all pre-defined time points. Samples foundwith non-detectable levels of viral DNA following dosing will not betested for levels of the transgene and will not be evaluated for latertime points.

Antibodies: Serum samples will be collected for analysis of levels ofantibodies to the adenovirus according to the Operation Manual.

Procedures for Handling Blood Samples: Refer to the Operation Manual forinstructions for a full description on Procedures for Safety Samples andProcedures for Q-PCR and Q-RT-PCR Determination.

Measurement of Effect

Tumor response will be assessed, using contrast and non-contrast brainmagnetic resonance imaging (MRI) with assessment based on the RANOcriteria, until progression of disease (local and central independentradiology review). For patients who do not progress or die, PFS will becensored at the time of initiation of alternative anticancer therapy,date of last radiologic assessment, or time of last contact.

Definitions

Response and progression will be evaluated in this study using the newinternational criteria proposed by the Response Assessment inNeuro-Oncology (RANO) Working Group [JCO, Updated Response AssessmentCriteria for High-Grade Gliomas: Response Assessment in Neuro-Oncology(RANO) Working Group, in press] Note: Lesions are either measurable ornon-measurable using the criteria provided below. The term “evaluable”in reference to measurability will not be used because it does notprovide additional meaning or accuracy.

Measurable Disease: Measurable disease is defined as bidimensionallycontrast-enhancing lesions with clearly-defined margins by MRI, with twoperpendicular diameters of at least 10 mm, visible on 2 or more axialslices which are preferably at most 5 mm apart with 0 mm skip. In theevent the MRI is performed with thicker slices, the size of a measurablelesion at baseline should be two times the slice thickness. In the eventthere are inter-slice gaps, this also needs to be considered indetermining the size of measurable lesions at baseline.

Measurement of tumor around a cyst or surgical cavity is problematic. Ingeneral, such lesions should be considered non-measurable unless thereis a nodular component measuring at least 10 mm in diameter. The cysticor surgical cavity should not be measured in determining response.

All tumor measurements must be recorded in millimeters (or decimalfractions of centimeters).

Non-measurable Disease: This is defined as either unidimensionallymeasurable lesions, masses with margins not clearly defined, or lesionswith maximal perpendicular diameters <10 mm.

Target Lesions: All measurable lesions up to a maximum of five lesionsshould be identified as target lesions and recorded and measured (sum ofthe products of the perpendicular diameters) at baseline. Target lesionsshould be selected on the basis of their size (lesions with the longestdiameters) and their suitability for accurate repeated measurements byimaging techniques. Occasionally, the largest lesions may not besuitable for reproducible measurements and the next largest lesionswhich can be measured reproducibly should be selected.

Non-target Lesions: For patients with recurrent disease who havemultiple lesions of which only one or two are increasing in size, theenlarging lesions should be considered the target lesions for evaluationof response. The other lesions will be considered non-target lesions andshould also be recorded.

Rarely, unequivocal progression of a non-target lesion requiringdiscontinuation of therapy, or development of a new contrast-enhancinglesion may occur even in the setting of stable disease (SD) or partialresponse (PR) in the target lesions. These changes would qualify asprogression.

Non-target lesions also include measurable lesions that exceed themaximum number of 5. Measurements of these lesions are not required butthe presence or absence of each should be noted throughout follow-up.

Guidelines for Evaluation of Measurable Disease

All measurements should be taken and recorded in metric notation using aruler or calipers. All baseline evaluations should be performed within72 hours before the beginning of the treatment.

Conventional MRI is required, CT is not acceptable. The same method ofassessment and the same technique should be used to characterize eachidentified and reported lesion at baseline and during follow-up.

These techniques should be performed with cuts of 10 mm or less in slicethickness contiguously. The MRIs will be evaluated both locally andcentrally by a core lab.

Response Criteria

Evaluation of Target Lesions: Complete Response (CR): Requires all ofthe following:

-   -   Complete disappearance of all enhancing measurable and        non-measurable disease sustained for at least 4 weeks    -   No new lesions    -   Stable or improved non-enhancing (T2/FLAIR) lesions    -   Patients must be off corticosteroids    -   Stable or improved clinically

Partial Response (PR): Requires all of the following:

-   -   ≧50% decrease compared to baseline in the sum of products of        perpendicular diameters of all measurable enhancing lesions        sustained for at least 4 weeks    -   No progression of non-measurable disease    -   No new lesions    -   Stable or improved non-enhancing (T2/FLAIR) lesions on same or        lower dose of corticosteroids compared to baseline scan    -   The corticosteroid dose at the time of the scan evaluation        should be no greater than the dose at time of the baseline scan    -   Stable or improved clinically

Stable Disease (SD): Requires all of the following:

-   -   Does not qualify for complete response, partial response, or        progression    -   Stable non-enhancing (T2/FLAIR) lesions on same or lower dose of        corticosteroids compared to baseline scan. In the event that the        corticosteroid dose has been increased, the last scan considered        to show stable disease will be the scan obtained when the        corticosteroid dose was equivalent to the baseline dose    -   Stable clinically

Progression: Defined by any of the following:

-   -   ≧25% increase in the sum of products of perpendicular diameters        of enhancing lesions compared to the smallest tumor measurement        obtained either at baseline (if no decrease) or best response,        on stable or increasing doses of corticosteroids    -   Significant increase in T2/FLAIR non-enhancing lesion on stable        or increasing doses of corticosteroids compared to baseline scan        or best response following initiation of therapy, not due to        co-morbid events (e.g. radiation therapy, demyelination,        ischemic injury, infection, seizures, post-operative changes, or        other treatment effects).    -   Any new lesion    -   Clear clinical deterioration not attributable to other causes        apart from the tumor (e.g. seizures, medication side effects,        complications of therapy, cerebrovascular events, infection,        etc.) or changes in corticosteroid dose    -   Failure to return for evaluation due to death or deteriorating        condition    -   Clear progression of non-measurable disease

If there is uncertainty regarding whether there is progression, thepatient may continue on treatment and remain under close observation. Ifsubsequent evaluations suggest that the patient is progressing, the dateof progression should be the time point at which this issue wasinitially raised.

These RANO Response Criteria are also summarized in the following table:

TABLE 6 Summary of the RANO Response Criteria CR PR SD PD# T1-Gd + None≧50% <50% ≧25% decrease decrease- increase* <25% increase T2/FLAIRStable or Stable or Stable or Increase* decrease decrease decrease NewLesion None None None Present* Corticosteroids None Stable or Stable orNA decrease decrease Clinical Stable or Stable or Stable or Decrease*Status increase increase increase Requirement All All All Any* forResponse CR = complete response; PR = partial response; SD = stabledisease; PD = progressive disease # Progression occurs when any of thecriteria with * is present NA: Increase in corticosteroids alone willnot be taken into account in determining progression in the absence ofpersistent clinical deterioration

Confirmatory Measurement/Duration of Response Confirmation

To be assigned a status of PR or CR, changes in tumor measurements mustbe confirmed by repeat assessments that should be performed 4 weeksafter the criteria for response are first met.

Duration of Overall Response

The duration of overall response is measured from the time measurementcriteria are met for CR or PR (whichever is first recorded) until thefirst date that recurrent or progressive disease is objectivelydocumented (taking as reference for progressive disease the smallestmeasurements recorded since the treatment started).

The duration of overall CR is measured from the time measurementcriteria are first met for CR until the first date that recurrentdisease is objectively documented.

Duration of Stable Disease

Stable disease is measured from the start of the treatment until thecriteria for progression are met, taking as reference the smallestmeasurements recorded since the treatment started.

Statistical Considerations Study Design

Objectives: To evaluate the safety, tolerability, and efficacy of singleand multiple doses of VB 111 (1×10¹², 3×10¹², 1×10¹³ viral particles[VPs]) in patients with recurrent GBM; to evaluate the distribution ofVB-111 after single and multiple IV infusions and the level ofantibodies to the adenovirus vector and transgene

Up to 90 subjects are anticipated to enroll in this study, with up to 29subjects at the 3×10¹² VP dose level, and up to 49 subjects at the1×10¹³ VP dose level. Cohorts 1-2 will enroll up to 6 patients in each.Cohort 3 will be conducted according to the Simon's 2 step method.

Step one will include the first 10 evaluable patients at the 1×10¹³ VPdose level or MTD.

For efficacy analysis, evaluable patients will be defined as those whoreceived at least one repeat dose of 1×10¹³ VPs or of MTD, or patientswho progressed sooner than 2 months after an initial dose of 1×10¹³ VPs(from cohort 3 or 4).

A subject will be considered to have a response if s/he is either aliveand progression free at 6 months or has at least a partial tumorresponse according to Rano criteria within 6 months post dosing. If lessthan 2 responses are observed in the step 1 subjects, step 2 will behalted, otherwise, an additional 19 subjects will enroll in step 2.

Cohort 4 will enroll up to 49 subjects.

It is anticipated that at least 29 patients enrolled in Cohorts 3 or 4will be evaluable after enrolling into Cohort 3-4 EXT. Based onhistorical information, it is anticipated that this sample will allowpreliminary assessment of safety and efficacy of this regimen for thetreatment of recurrent GBM.

The following study stopping rules for halting the study will beapplied:

-   -   If 3 out of 6 subjects in the Cohorts 1 & 2 experience drug        related DLT (or 5 out of 9 or 6 out of 12);    -   If 2 of the subjects in Cohort 1 experience a DLT;    -   If ANY death occurs within two weeks after the product is given,        except death due to disease progression or clearly unrelated to        study drug. Enrollment will be temporarily suspended for an ad        hoc, emergency IDMC meeting to review the case and make a        recommendation if enrolment can be reinstated.

Safety and Tolerability Endpoints

To evaluate the safety and tolerability of single and multiple doses ofVB-111 (1×10¹², 3×10¹², 1×10¹³ VPs) the following safety endpoints willbe assessed throughout the study:

-   -   Adverse events will be recorded on an ongoing basis for the        whole duration of the study. Adverse events will be assessed for        seriousness, relatedness to study drug, and severity (According        to CTCAE 4.0).    -   Vital signs will be recorded at screening, prior to each dosing,        30 and 60 minutes, 4 and 6 hours after the first dosing and at        all follow up visits.    -   Physical examinations will be conducted in Cohorts 1-2 at        screening, baseline, Days 14, 28, 56, 84, 112, 140 and Day 168.        In Cohorts 3-4, a physical exam will be conducted at        screening/baseline, before each dosing, and the study        completion/early termination.    -   12-lead ECG will be obtained in Cohorts 1-2 at screening, prior        to dosing, Day 28 and Day 168 (or ET). In Cohorts 3-4, an ECG        will be obtained at screening/baseline, the Day 56 visit, and        the study completion/early termination visit.    -   Safety laboratory assessment (blood hematology, chemistry, and        urinalysis) will be conducted at screening, prior to dosing, and        at all patient visits.

Primary Efficacy Endpoint: To evaluate the efficacy of single ormultiple doses of VB-111 (1×10¹², 3×10¹², 1×10¹³ VPs) in recurrent GBMsubjects, 6 months progression free survival (PFS) will be assessed,defined as proportion of subjects who are progression free at 6 monthsfrom enrollment. In another aspect, overall survival will be assessed asthe primary efficacy endpoint and 6-months PFS will be assessed as asecondary efficacy endpoint. Overall survival is defined as the timefrom enrollment until death from any cause. Patients will be followedfor survival status after completion or removal from the study forprogression or toxicity, and will be included in the PFS and OSanalyses.

Secondary Efficacy Endpoints: To evaluate the efficacy of single ormultiple doses of VB-111 (1×10¹², 3×10¹², 1×10¹³ VPs), or multiple dosesof VB-111 together with bevacizumab in recurrent GBM subjects thefollowing secondary efficacy endpoints will be assessed:

-   -   In one aspect, secondary efficacy endpoints will be assessed by        Overall survival (OS): Overall survival is defined as the time        from enrollment until death from any cause. Patients will be        followed for survival status after completion or removal from        the study for progression or toxicity, and will be included in        the PFS and OS analyses.    -   In another aspect, secondary efficacy endpoints will be assessed        by Progression Free Survival (PFS): Progression free survival is        defined as the time from enrollment until objective tumor        progression, assessed according to Response Assessment in        Neuro-Oncology (RANO) Working Group (see details in section        8.3.1).    -   Progression Free Survival at 6 months (PFS-6) is defined as the        proportion of subjects who are progression free at 6 months from        enrollment    -   Event free survival (EFS): Event-free survival is defined as        measurement from the date of enrollment until termination of        treatment due to toxicity, disease progression, relapse, or        death from any cause.    -   Tumor response: based on MRI, assessed according to Response        Assessment in Neuro-Oncology (RANO) Working Group (see details        in section 8.3.1)

Power and Sample Size Considerations

In one embodiment, the primary efficacy endpoint is Overall Survival.Using data presented in Friedman et al., “Bevacizumab alone and incombination with irinotecan in recurrent glioblastoma,” J. Clin Oncol.27(28):4733-40 (2009), OS in the bevacizumub group at 12 months is 25%.Based on preliminary data, it is expected that the 12 month overallsurvival rate in the VB-111 group will be approximately 50% (hazardratio=0.5). Assuming a proportional hazards model, a sample size of 45patients in the VB-111 group generating approximately 23 events, a 0.05level two-sided Logrank test will have 80% power to detect a differenceof survival curves. The final analysis will compare Kaplan Meiersurvival curves of BEV (based on Friedman et al data) vs. VB-111survival data using the Log-rank Test (Piantadosi 2005).

Alternatively, this study will assess efficacy endpoints using atwo-stage Simon design. In this study response is defined by either 6months PFS or at least a partial tumor response. With a minimum of 10patients and a maximum of 29 patients the null hypothesis that the trueproportion of PFS-6 is at most 10% vs the alternative hypothesis thatthe 6 month PFS is at least 30%:

H0: PFS-6<=10% H1: PFS-6 >=30%

This has a significance level of 5% when the true proportion is 10% and80% power when the true proportion is at least 30%.

Unless toxicity is encountered or the study is stopped at the interimanalysis, this study anticipates accruing between 3-6 subjects in Cohort1, 3-6 subjects in Cohort 2, up to 29 subjects in Cohort 3 and up to 49subjects in Cohort 4.

It is anticipated that at least 29 patients enrolled in Cohorts 3 or 4will be evaluable after enrolling into Cohort 3-4 EXT. Based onhistorical information, it is anticipated that this sample will allowpreliminary assessment of safety and efficacy of this regimen for thetreatment of recurrent GBM.

Statistical Methods

All data collected will be summarized and presented. Continuousvariables will be described as the mean, median, standard deviation andrange of n observations. Categorical data will be described withcontingency tables including frequency and percent. Individual subjectlistings will be generated and presented.

Confidence intervals will be calculated at the (two-sided) 95% level ofconfidence (except for the primary efficacy and safety endpoints, whichwill be 99% and 95% (one-sided) respectively.

All patients meeting the eligibility criteria who have signed a consentform and have received VB-111 will be considered evaluable for safetyanalysis.

Statistical descriptions and analyses will be carried out using SASstatistical analysis software (SAS Institute, Inc., Cary, N.C., USA).

Demographic Parameters

Demographic parameters including sex, age, height and weight will besummarized overall and by treatment group.

Safety and Toxicity Analyses

Adverse events monitoring and clinical findings will be used to assesssafety.

AEs will be categorized by SOC and Preferred Terms using the MedDRAdictionary. The incidence of AEs as well as the severity andrelationship to study drug will be presented. As per NCI CTCAE Version4.0, the term toxicity is defined as adverse events that are classifiedas either possibly, probably, or definitely related to study treatment.The maximum grade for each type of toxicity will be recorded for eachpatient and will be used for reporting. Frequency tables will bereviewed to determine toxicity patterns. In addition, all adverse eventdata graded as 3, 4, or 5 and classified as either “unrelated orunlikely to be related” to study treatment in the event of an actualrelationship developing will be reviewed.

Serious adverse events will be similarly summarized.

Infusional adverse events will also be summarized by both theinvestigator's and the sponsor's assessment of causality.

The number and proportion of patients who have one or more AEs will bepresented as well.

Clinical findings will include evaluation of physical examinations,vital signs and laboratory test results, concomitant medications, andwithdrawals/terminations. These findings will be summarized by dosegroup. Continuous variables will be described as the mean, median,standard deviation, and range of n observations. Categorical data willbe described with contingency tables including frequency and percent.

Efficacy Analyses

Overall survival (OS) is defined as the time from the first date ofstudy treatment until the date of subject death from any cause. Forsubjects who have not died, survival data will be censored at thesubject's last known alive date. The Kaplan-Meier method will be used toestimate the distribution and median OS for subjects treated at the MTDlevel.

Progression Free Survival (PFS) is defined as the time from the firstdate of study treatment to documented disease progression, or to deathfrom any cause whichever occurs earlier. Tumor response and diseaseprogression will be assessed by the investigator according to RANOcriteria. The proportion of subjects who have 6 months PFS will bepresented for each treatment group. For patients who do not progress ordie, PFS will be censored at the time of initiation of alternativeanticancer therapy, date of last tumor assessment, or time of lastcontact. The Kaplan-Meier method will be used to estimate thedistribution and median PFS for subjects treated at the 3×10¹² and1×10¹³ VPs levels, or multiple doses of VB-111 together withbevacizumab.

Event Free Survival is defined as measurement from the date ofenrollment until termination of treatment due to toxicity, diseaseprogression, relapse, or death from any cause. The Kaplan-Meier methodwill be used to estimate the distribution and median of event freesurvival for subjects treated at the MTD level.

Tumor Response is defined according the RANO Response criteria. SeeSection 8.3 for definitions of Complete Response, Partial Response,Stable Disease and Progression. Tumor response will be assessed atScreening, prior to dosing, Days 14, 28, 56, 84, 112, 140 (Cohorts 1-2only) and 168 and every 2 months thereafter, using contrast andnon-contrast brain magnetic resonance imaging (MRI). The frequency andpercentage of tumor response will be presented by timepoint andtreatment group. For Cohorts 3-4, and the extension, tumor response willbe assessed at Screening, prior to dosing, Days 28, 56, 84, 112, andevery 2 months thereafter. Following this protocol amendment (Version7.0), scans will be performed every 2 months from initial dose.

Biodistribution

The Biodistribution of VB-111 in blood will be presented.

Number of Patients Planned to be Enrolled

Based on the study design it is estimated that a maximum of 90 eligible(3-6 in each of cohorts 1 and 2, up to 29 in cohort 3, and up to 49 inCohort 4) with recurrent GBM cancer are needed, 29 evaluable patientsare required for efficacy assessment.

Criteria for Termination of the Trial

The sponsor reserves the right to terminate the study early foradministrative (e.g. center not complying with GCP) or safety reasons asdescribed under 4.2.

Procedure for Accounting for Missing, Unused and Spurious Data

Missing data will be indicated in the listings, but excluded from alldescriptive analyses. All data will be listed, including otherwiseunused data. Spurious data will be identified as such, whereverpossible.

Selection of Patients to be Included in the Analyses

The Full Analysis Set will consist of all patients enrolled who receivestudy medication. The biodistribution subset will consist of allpatients for whom a VB-111 profile is obtained.

Interim Analyses:

An interim analysis will be performed after enrollment of 10 evaluablepatients (as defined above) who have reached day 168 time point (6months) or have discontinued prior to that, The results of the interimanalysis will determine whether the remainder of the patients will beenrolled into cohort 4 in order to complete the study.

Example 3

Methods:

VB-111 was administered as a single intravenous infusion at escalatingdoses from 1×10¹² (cohort 1) to 3×10¹² (cohort 2) viral particles (VPs),followed by repeat doses of 3×10¹² or 1×10¹³ every 2 months (cohorts3-4). Assessments included safety, pharmacokinetics, tumor responseaccording to RANO criteria and overall survival.

Results:

Twenty eight patients aged 26-74 years at 3 medical centers in the USreceived up to 8 repeat doses of VB-111. The median overall survival was360 [range: 70-574] and 266 days [range: 28-664] for patients receivingat least one dose of 1×10¹³ VPs (high dose) vs subjects who receivedlower doses, respectively (p NS). Progression free survival was 87 vs 55days for patients who received high dose and for lower doses,respectively (p=0.01). Median follow-up was 232 days. Three patients hada partial response (PR) at 82, 86 and 408 days post initial VB-111dosing. Twenty one of the patients who progressed after VB-111 treatmentreceived bevacizumab off study; 7 of the 15 evaluable patients (47%) hada PR compared to a 30% expected PR rate according to literature. VB-111was safe and well tolerated, 53 adverse events were reported, 14 wereclassified as possibly related to VB-111. All events were of CTCAE grade1-2 except one grade 3 pulmonary embolism (PE). There were no studyrelated deaths. One patient developed peri-tumoral edema post dosing,which resolved with corticosteroid therapy. Events occurring in morethan 10% of the patients included headache and fatigue.

As this study continued, additional subjects with progressive diseasewere treated with bevacizumab and the study data were recalculatedaccordingly. Twenty three of the subjects who had progressive disease(PD) on VB-111 treatment received bevacizumab off study; 6 of the 15evaluable subjects (40%) had a partial response (PR) compared to 30%expected according to literature. The data are provided in the Tablebelow.

TABLE 7 Post-VB-111 Response to bevacizumab Exposure to Subject IDbevacizumab Best Response* 60-001 Yes PD 60-002 Yes PD 60-003 Yes SD60-004 Yes SD 60-005 Yes PR 61-001 Yes Unknown 61-002 Yes Unknown 61-003Yes SD 61-004 Yes Unknown 61-005 Yes Unknown 62-002 No N/A 62-003 Yes PR62-004 Yes PR 62-005 Yes Unknown 62-006 Yes SD 62-007 Yes PD 62-008Unknown Unknown 62-009 Yes Unknown 62-010 Subject still stable on VB-11162-011 Yes PD 62-012 Yes Unknown 62-013 Yes PR 62-014 Unknown Unknown62-015 Yes SD 62-016 Yes PR 62-017 Yes PR 62-018 Yes Unknown 62-019Subject still stable on VB-111 *PD, progressive disease; PR, partialresponse; SD, stable disease

Additional patients were subsequently added to the study and the datawere recalculated accordingly. Forty six patients aged 27-76 years at 4medical centers in the US and Israel received up to 13 repeat doses ofVB-111. Of these, 30 patients received the high dose (1×10¹³ VPs). Therewere 22 related adverse events, 19 CTCAE grade 1-2; grade 3 includedpulmonary embolism, peri-tumoral edema and DVT. The median overallsurvival was 360 [range: 70-574] and 266 days [range: 28-664] forpatients receiving at least one high dose vs. subjects who receivedlower doses, respectively (p NS). Progression free survival was 63 vs.55 days for patients who received high vs. lower doses, respectively(p=0.01). Median follow-up was 232 days. Six patients had a partialresponse and/or prolonged disease stability (≧180 days). Tumor growthrates showed a statistically significant dose response. Eleven patientsreceived combination therapy of up to 4 doses of VB-111 together withbevacizumab after progression on VB-111 monotherapy. Median time tosecond progression was 93 days. These data are shown in FIG. 2. FIG. 3provides an example of a patient who was treated with a combination ofVB-111 and bevacizumab upon disease progression. VB-111 was safe andwell tolerated both as monotherapy and combined therapy.

Conclusions:

VB-111 was safe and well tolerated in patients with recurrent GBM withrepeat doses of up to 1×10¹³ VPs. Tumor responses were seen. Overallsurvival was about 3 months longer compared to historical data inrecurrent GBM including standard of care anti-angiogenic agents. Datasuggests that VB-111 potentiates the response to bevacizumab given atfurther progression.

Example 4

Administration of Multiple Doses of Ad5-PPE-1-3×-Fas-c Combined withBevacizumab

OBJECTIVE: To evaluate the safety, tolerability, and efficacy ofmultiple doses of VB-111 1×10¹³ viral particles (VP) in patients withrecurrent GBM every 8 weeks as monotherapy, combined with bevacizumab 10mg/Kg every 2 weeks upon progression compared to bevacizumab monotherapyin patients with recurrent GBM.

The patients in the intervention arm will first receive VB-111, followedby combination of VB-111 with bevacizumab upon progression.

In one embodiment, further efficacy of VB-111 can be observed incombination with bevacizumab even after progression on VB-111monotherapy, since synergism may potentiate VB-111 activity and overcomedrug resistance. Also, in some cases delayed response to VB-111 wasobserved in the patients several months after receiving VB-111. Thus,some patients may experience rapid disease progression upon studyenrollment without the opportunity to respond to VB-111. The proposedregimen may provide benefit from such delayed responses.

Inclusion Criteria

First or second progression or first recurrence of Glioblastoma(according to updated RANO Criteria, see Table 8) following standard ofcare treatment with temozolomide and radiation;

A histologically confirmed diagnosis of GBM. Patients with surgicallyresectable disease at recurrence may be enrolled, even if there is noresidual disease upon surgical resection. An interval of at least 4weeks between prior surgical resection and study enrollment;

TABLE 8 Criteria for Response Assessment Incorporating MRI and ClinicalFactors (From updated RANO criteria as published in Wen et al., “Updatedresponse assessment criteria for high-grade gliomas: response assessmentin neuro-oncology working group., J Clin Oncol 28: 1963-1972 (2010).Response Criteria Complete Requires all of the following: completedisappearance of all response enhancing measurable and non-measurabledisease sustained for at least 4 weeks; no new lesions; stable orimproved non- enhancing (T2/FLAIR) lesions; patients must be offcorticosteroids (or on physiologic replacement doses only); and stableor improved clinically. Note: Patients with non- measurable disease onlycannot have a complete response; the best response possible is stabledisease. Partial Requires all of the following: ≧50% decrease comparedwith response baseline in the sum of products of perpendicular diametersof all measurable enhancing lesions sustained for at least 4 weeks; noprogression of non-measurable disease; no new lesions; stable orimproved non-enhancing (T2/FLAIR) lesions on same or lower dose ofcorticosteroids compared with baseline scan; the corticosteroid dose atthe time of the scan evaluation should be no greater than the dose attime of baseline scan; and stable or improved clinically. Note: Patientswith non-measurable disease only cannot have a partial response; thebest response possible is stable disease. Stable Requires all of thefollowing: does not qualify for complete disease response, partialresponse, or progression; stable non- enhancing (T2/FLAIR) lesions onsame or lower dose of corticosteroids compared with baseline scan. Inthe event that the corticosteroid dose was increased for new symptomsand signs without confirmation of disease progression on neuroimaging,and subsequent follow-up imaging shows that this increase incorticosteroids was required because of disease progression, the lastscan considered to show stable disease will be the scan obtained whenthe corticosteroid dose was equivalent to the baseline dose. Progres-Defined by any of the following: ≧25% increase in sum of the sionproducts of perpendicular diameters of enhancing lesions compared withthe smallest tumor measurement obtained either at baseline (if nodecrease) or best response, on stable or increasing doses ofcorticosteroids*; significant increase in T2/FLAIR non-enhancing lesionon stable or increasing doses of corticosteroids compared with baselinescan or best response after initiation of therapy* not caused bycomorbid events (e.g., radiation therapy, demyelination, ischemicinjury, infection, seizures, postoperative changes, or other treatmenteffects); any new lesion; clear clinical deterioration not attributableto other causes apart from the tumor (e.g., seizures, medication adverseeffects, complications of therapy, cerebrovascular events, infection,and so on) or changes in corticosteroid dose; failure to return forevaluation as a result of death or deteriorating condition; or clearprogression of non-measurable disease.

All measurable and non-measurable lesions must be assessed using thesame techniques as at baseline. Abbreviations: MRI, magnetic resonanceimaging; FLAIR, fluid-attenuated inversion recovery. * Stable doses ofcorticosteroids include patients not on corticosteroids

Exclusion Criteria

Pregnant or breastfeeding patients;

Evidence of CNS haemorrhage CTCAE grade 2 or above on baseline MRI;

Patients who suffered from an acute cardiac event within the last 12months;

Patients with active vascular disease, either myocardial or peripheral(i.e. acute coronary syndrome, cerebral stroke, transient ischemicattack or arterial thrombosis or symptomatic peripheral vascular diseasewithin the past 3 months);

Patients with known proliferative and/or vascular retinopathy;

Patients with known liver disease (alcoholic, drug/toxin induced,genetic, or autoimmune);

Patients that have undergone major surgery within the last 4 weeksbefore enrollment;

Minor surgical procedure, e.g. stereotactic biopsy, within 7 days offirst study treatment; placement of a vascular access device, within 2days of first study treatment;

History of intracranial abscess within 6 months prior to first studytreatment;

History of active gastrointestinal bleeding within 6 months prior tofirst study treatment;

Serious, non-healing wound, active ulcer, or untreated bone fracture;

Treatment Plan

This study is a prospective, randomized, controlled, 2-arm, open label,multi-center, Phase 3 study, measuring the efficacy, safety,tolerability of multiple doses of intravenously administered VB-111followed by VB-111 and bevacizumab as compared to bevacizumabmonotherapy in patients with recurrent GBM.

Patients will be screened for eligibility and then, up to 28 days later,at the baseline visit, randomized to one of two treatment groups in a1:1 ratio (investigative arm to control arm). Patients will berandomized and stratified according to age at randomization (≦60 years,<60 years), KPS (<80, ≧80) and progression (1st progression, 2ndprogression). The intervention arm will receive monotherapy with VB-111administered as multiple intravenous infusions of 1×10¹³ VPs every 8weeks, which will, upon progression, be combined with bevacizumab 10mg/kg every 2 weeks. This regimen will continue until furtherprogression. The control arm will receive monotherapy with bevacizumab10 mg/kg every 2 weeks until progression.

The patients will remain in the study until discontinuation due todisease progression or withdrawal. Thereafter, follow-up for survivalwill continue until the patient expires.

An overview of the study is shown in FIG. 1.

Study Visits Screening, Day −28-0 (All Patients)

Prospective patients will be screened within 4 weeks of enrollment.Patients will be informed of the advantages, risks and constraints ofthe study and will be asked to sign an informed consent form. Patientswho have provided consent will be assigned an identification numberwhich consists of the center number (e.g. 01) plus the subject/patientnumber in sequential order (e.g., 001, 002, 003, etc.). The fullidentification number for the first patient at center 01 will be 01-001,01-002, etc.

Patients will proceed to screening assessments as shown in Example 2.

Baseline Visit Day 1 (All Patients)

Study treatment (VB-111 or bevacizumab) will be administered as anoutpatient.

Once the subject/patient is found eligible, he/she will be scheduled toarrive at the clinic within 4 weeks from screening in a fasting stateuntil 30 minutes following study drug administration.

Prior to dosing (Dl):

On the day of admission to the site, each subject/patient will bere-verified for eligibility according to inclusion/exclusion criteriaand then tested within 24 hours prior to dosing for the followingevaluations:

ADVERSE EVENTS: Patients will be asked about changes in their physicaland mental status since signing the consent form.

CONCOMITANT MEDICATIONS: Patients will be asked about changes in theirtherapies since signing the consent form.

TUMOR MEASURE: Contrast and non-contrast brain MRI imaging will be doneto assess extent of cancer. If there are less than 2 weeks betweenscreening and baseline visits, only 1 MRI will be required.

Laboratory Analyses:

Blood Samples:

-   -   Hematology: hemoglobin, complete blood count with differential,        INR, PT and activated PTT;    -   Chemistry: electrolytes, creatinine and blood urea nitrogen,        bilirubin, alkaline phosphatase, ALT and AST, calcium, total        protein and albumin;    -   Antibody to the Ad-5 virus sample.

Urine will be collected for the following evaluations:

-   -   Routine urinalysis    -   Dipstick for Proteinuria        -   No dose modifications for grade 1/2 events        -   Grade 3-(UPC>3.5, urine collection >3.5 g/24 hr, or dipstick            4+): Hold bevacizumab treatment until ≦Grade 2, as            determined by either UPC ratio ≦3.5 or 24 hr collection ≦3.5            g        -   Grade 4 (nephrotic syndrome): Discontinue bevacizumab

HEALTH-RELATED QUALITY OF LIFE ASSESSMENTS: EORTC core Quality of LifeQuestionnaire (QLQ-C30) and a Brain Cancer Module (BCM20) will becompleted prior to receiving treatment.

RANDOMIZATION: Patients meeting all of the entry criteria will berandomized to one of two treatment groups in a 1:1 ratio, respectively,using a centralized randomization procedure: (1) VB-111 1×10¹³ VPsmonotherapy every 8 weeks followed by, upon progression, VB-111 every 8weeks and bevacizumab 10 mg/kg every 2 weeks or (2) Bevacizumabmonotherapy: 10 mg/kg every 2 weeks. Randomization will be stratifiedaccording to age at randomization (≦60 years, <60 years), KPS (<80, ≧80)and progression (1^(st) progression, 2^(nd) progression).

VITAL SIGNS: Vital signs (supine, systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded 15 minutes prior to dosing.

PROPHYLACTIC TREATMENTS: VB-111 RANDOMIZED PATIENTS: For patientsrandomized to the VB-111 treatment arm, the following prophylactictreatments will be administered:

ANTI-PYRETIC TREATMENT: To avoid fever following study drugadministration, prior to treatment with VB-111 only all patients willreceive 1000 mg of acetaminophen starting 1-2 hours prior to dosingfollowed by 500 mg PRN 24 hours.

CORTICOSTEROID TREATMENT: To reduce potential edema response during drugadministration, dexamethasone treatment will be administered prior totreatment with VB-111 only:

Initial dose with VB-111: 10 mg will be administered 30 minutes prior todosing, followed by 4 mg×2/day for 14 days post dosing.

Subsequent doses with VB-111: 10 mg will be administered 30 minutesprior to dosing, followed by 4 mg×2/day for 3 days post dosing. Furthercorticosteroid treatment will be administered at Investigator'sdiscretion.

If patients begin the study already on steroids, all efforts should bemade by the Investigator not to change the steroid dose within 5 days ofdisease assessment, unless clinically warranted. A decision to continuethe steroids or to begin tapering after this period of time is at thediscretion of the Investigator.

Study Drug Administration:

VB-111: Prior to infusion, the solution for injection should be broughtto room temperature. Maximum time for drug in saline is 90 minutes atroom temperature (60 minutes plus a 30 minute window). The vials shouldbe opened in a biological safety cabinet and combined with saline (seeVB-111 Infusion Preparation below). VB-111 will be infused to thepatient at the relevant dosage according to the patient's weight,according to the Operations Manual. Specifically, for patients who weighless than 50 Kg, the dose will be reduced by 30%. A single infusion ofVB-111 should be administered at 3 ml/minute. Patients receiving VB-111should remain in the clinic for 8 hours for observation.

BEVACIZUMAB: To be administered as standard of care (SOC).

VITAL SIGNS: VB-111 RANDOMIZED PATIENTS: The vital signs (systolic anddiastolic blood pressure, peripheral heart rate, body temperature,respiration rate) will be recorded at 30 and 60 minutes after dosing andat 4 and 6 hours post dosing, and/or upon disappearance of any adverseevent, whichever comes first.

ADVERSE EVENTS: Patients will be monitored for adverse events throughtheir observation following treatment at the clinic.

CONCOMITANT MEDICATIONS: Any concomitant medications administered duringthe patients' observation following treatment should be recorded.

VB-111 Monotherapy Follow Up Visits Safety Telephone Contact Every OtherMonth in Between Dosing Visits (Days 28, 84, 140, etc.)

A telephone call should be made every other month in between dosingvisits (days 28, 84, 140, etc.) to the subject/patient to inquire aboutAEs and changes in medication.

Further VB-111 Administration (Every 8 Weeks Following Initial Dose: Day56, Day 112, Day 168, etc.)

On the day of admission to the site, each subject/patient will bere-verified for eligibility according to inclusion/exclusion criteria(where applicable) and then tested within 24 hours prior to dosing.Patients without evidence of progressive disease will be considered forfurther dosing. This visit and VB-111 dosing will be repeated every 8weeks until disease progression.

The following assessments will be performed:

ADVERSE EVENTS: Patients will be asked about changes in their physicaland mental status since the last visit.

CONCOMITANT MEDICATIONS: Patients will be asked about changes in theirtherapies since the last visit.

HEALTH-RELATED QUALITY OF LIFE ASSESSMENTS: EORTC core

Quality of Life Questionnaire (QLQ-C30) and a Brain Cancer Module(BCM20) will be completed prior to receiving treatment.

TUMOR MEASURE: Patients will be assessed for response using contrast andnon-contrast brain magnetic resonance imaging (MRI) with assessmentbased on the RANO criteria. MRI imaging will be done up to 72 hoursprior to dosing to assess the extent of cancer. (If the patient hasconfirmed disease progression, the patient should be considered forparticipation in the combination therapy with VB-111 and bevacizumab.VB-111 1×10¹³ VPs will be administered every 8 weeks and bevacizumabwill be administered every 2 weeks.)

PHYSICAL EXAM: Physical Examination will focus on tumor organs and tumormeasurements, according to RANO criteria, as well as the following: Headand neck; Eyes; Lungs; Heart; Abdomen; Joints; Peripheral circulation;Skin; or Neurologic.

ECG (Day 168 and Early Termination Only): A standard 12-leadelectrocardiogram with rhythm strip should be performed.

Laboratory Analyses:

Blood samples will be collected for the following evaluations:

-   -   Hematology: hemoglobin, complete blood count with differential,        INR, PT and activated PTT;    -   Chemistry: electrolytes, creatinine and blood urea nitrogen,        bilirubin, alkaline phosphatase, ALT and AST, calcium, total        protein and albumin;    -   Serum pregnancy test    -   Antibody to the Ad-5 virus sample.

Urine will be collected for the following evaluations:

-   -   Routine urinalysis    -   Dipstick for Proteinuria    -   No dose modifications for grade 1/2 events    -   Grade 3-(UPC>3.5, urine collection >3.5 g/24 hr, or dipstick        4+): Hold bevacizumab treatment until ≦Grade 2, as determined by        either UPC ratio ≦3.5 or 24 hr collection ≦3.5 g    -   Grade 4 (nephrotic syndrome): Discontinue bevacizumab

VITAL SIGNS: Vital signs (supine, systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded 15 minutes prior to dosing.

ANTI-PYRETIC TREATMENT: To avoid fever following study drugadministration, prior to treatment with VB-111 all patients will receive1000 mg of acetaminophen 1-2 hours prior to dosing followed by 500 mgevery 4 hours for 24 hours.

CORTICOSTEROID TREATMENT: To reduce potential edema response during drugadministration, prior to treatment with VB-111 dexamethasone treatmentwill be administered: 10 mg will be administered 30 minutes prior todosing, followed by 4 mg×2/day for 3 days post dosing. Furthercorticosteroid treatment will be administered at Investigator'sdiscretion.

If the patient is on steroids at the time of dosing, all efforts shouldbe made by the Investigator not to change the steroid dose within 5 daysof disease assessment, unless clinically warranted. A decision tocontinue the steroids or to begin tapering after this period of time isat the discretion of the Investigator.

STUDY DRUG ADMINISTRATION: Infusion will be done according to theoperation manual. Prior to infusion, the saline should be brought toroom temperature. The vials should be opened in a biological safetycabinet and injected into normal saline for infusion according to theoperation manual (see VB-111 Infusion Preparation) below. The finalsolution for administration should be administrated not more than 90minutes after preparation (60 minutes plus a 30 minute window). VB-111will be infused to the patient at the relevant dosage according to thepatient's weight, as detailed in the Operations Manual. For patientsless than 50 Kg of weight, the dose of 1×10¹³ VPs will be reduced by30%. The intravenous infusions of diluted VB-111 should be administeredat 3 mL/minute

A regular meal will be allowed 0.5 hour after dosing.

ADVERSE EVENTS: Patients will be monitored for adverse events throughtheir observation following treatment at the clinic.

CONCOMITANT MEDICATIONS: Any concomitant medications administered duringthe patients' observation following treatment should be recorded.

End of VB-111 Monotherapy Upon Disease Progression

Patients will continue to receive VB-111 every 8 weeks provided thatthey continue to be stable. Once patients experience diseaseprogression, they will return to the clinic for a final study visit. Inaddition, if patients withdraw consent or it is determined by theInvestigator that the patient should not continue in the study, thepatient will return for an Early Termination Visit. Patients will becontacted by telephone every 2-3 months to follow up on survival.

VB-111+Bevacizumab Administration as Combination Treatment

At the time the patient experiences disease progression, thesubject/patient will initiate treatment with VB-111 and bevacizumab as acombination therapy. VB-111 1×10¹³ VPs will be administered every 8weeks and bevacizumab will be administered every 2 weeks.

Since a minimum of 8 weeks is required between doses of VB-111, if asubject/patient progresses before 8 weeks, the subject will begintreatment with bevacizumab (10 mg/kg) every 2 weeks and at 8 weeks fromthe last dose of VB-111, the subject/patient will receive the firstcombination dose with VB-111.

Patients will be re-informed of the advantages, risks and constraints ofthe combination therapy. The following assessments will be performed oneach day of combination treatment:

ADVERSE EVENTS: Patients will be asked about changes in their physicaland mental status since the last visit.

CONCOMITANT MEDICATIONS: Patients will be asked about changes in theirtherapies since the last visit.

HEALTH-RELATED QUALITY OF LIFE AND NEUROCOGNITIVE FUNCTION: EORTC coreQuality of Life Questionnaire (QLQ-C30) and a Brain Cancer Module(BCM20) will be completed prior to receiving treatment.

TUMOR MEASURE: Patients will be assessed for response using contrast andnon-contrast brain magnetic resonance imaging (MRI) with assessmentbased on the RANO criteria. MRI imaging will be done up to 72 hoursprior to dosing to assess extent of cancer.

PHYSICAL EXAM: Physical Examination will focus on tumor organs and tumormeasurements, according to RANO criteria, as well as the following: Headand neck; Eyes; Lungs; Heart; Abdomen; Joints; Peripheral circulation;Skin; or Neurologic

VITAL SIGNS: Vital signs (supine, systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded 15 minutes prior to dosing.

ECG (to be performed every 6 months from the initial combination dose):A standard 12-lead electrocardiogram with rhythm strip should beperformed.

Laboratory Analyses:

Blood sample will be collected for the following evaluations:

-   -   Hematology: hemoglobin, complete blood count with differential,        INR, PT and activated PTT;    -   Chemistry: electrolytes, creatinine and blood urea nitrogen,        bilirubin, alkaline phosphatase, ALT and AST, calcium, total        protein and albumin;    -   Serum pregnancy test;    -   Antibody to the Ad-5 virus sample.

Urine will be collected for the following evaluations:

-   -   Routine urine analysis    -   Dipstick for Proteinuria        -   No dose modifications for grade 1/2 events        -   Grade 3-(UPC>3.5, urine collection >3.5 g/24 hr, or dipstick            4+): Hold bevacizumab treatment until <Grade 2, as            determined by either UPC ratio ≦3.5 or 24 hr collection ≦3.5            g    -   Grade 4 (nephrotic syndrome): Discontinue bevacizumab

ANTI-PYRETIC TREATMENT: To avoid fever following study drugadministration, prior to treatment with VB-111 all patients will receive1000 mg of acetaminophen 1-2 hours prior to dosing followed by 500 mgevery 4 hours for 24 hours.

CORTICOSTEROID TREATMENT: To reduce potential edema response during drugadministration, prior to treatment with VB-111 dexamethasone treatmentwill be administered: 10 mg will be administered 30 minutes prior todosing, followed by 4 mg×2/day for 3 days post dosing. Furthercorticosteroid treatment will be administered at Investigator'sdiscretion.

If the patient is on steroids at the time of dosing, all efforts shouldbe made by the Investigator not to change the steroid dose within 5 daysof disease assessment, unless clinically warranted. A decision tocontinue the steroids or to begin tapering after this period of time isat the discretion of the Investigator.

BEVACIZUMAB ADMINISTRATION: Bevacizumab will be administered by infusionat a dose of 10 mg/kg before VB-111 on dosing days. The rate of infusionshall be according to the package insert for bevacizumab: Bevacizumabshould be delivered over 60 minutes (+30 minute window) as an IVinfusion. If the 60-minute infusion is tolerated, all subsequentinfusions may be administered over 30 minutes.

VB-111 ADMINISTRATION: Infusion will be done according to the operationmanual. Prior to infusion, the saline should be brought to roomtemperature. The vials should be opened in a biological safety cabinetand injected into normal saline for infusion according to the operationmanual (see VB-111 Infusion Preparation below). The final solution foradministration should be administrated not more than 90 minutes afterpreparation (60 minutes plus a 30 minute window). VB-111 will be infusedto the patient at the relevant dosage according to the patient's weight,as detailed in the Operations Manual. For patients less than 50 Kg ofweight, the dose of 1×10¹³ VPs will be reduced by 30%. The intravenousinfusions of diluted VB-111 should be administered at 3 mL/minute.

VITAL SIGNS: For the initial combination dose, vital signs (systolic anddiastolic blood pressure, peripheral heart rate, body temperature,respiration rate) will be recorded at 30 and 60 minutes after dosing andat 4 and 6 hours post dosing, and/or upon disappearance of any adverseevent, whichever comes first. For further doses, vital signs should berecorded 15 minutes prior to dosing and 30 minutes following dosing

ADVERSE EVENTS: Patients will be monitored for adverse events throughtheir observation following treatment at the clinic.

CONCOMITANT MEDICATIONS: Any concomitant medications administered duringthe patients' observation following treatment should be recorded.

Follow Up: Combination Treatment with VB-111 and Bevacizumab:Bi-Weekly Treatment with Bevacizumab

Bevacizumab will be administered according to standard of care practicesevery 2 weeks. It is preferred that the subject/patient visit the studyclinic to receive this treatment and be seen by the Investigator toassess safety (via collection of adverse event and concomitantmedication information). However, if the subject/patient lives more than1 hour commute from the clinic, the subject/patient may receive thistreatment from his/her local oncologist and safety information(collection of adverse event and concomitant medication information)should be transferred to the study clinic for inclusion into studyrecords. Local visits should be pre-approved by the Medical Monitor on aper patient basis.

Days Following Initial Combination Treatment

One month following the initial combination treatment, each patient willbe required to return to the clinic in a fasting state, for thefollowing evaluations:

ADVERSE EVENTS: Patients will be asked about changes in their physicaland mental status since the last visit.

CONCOMITANT MEDICATIONS: Patients will be asked about changes in theirtherapies since the last visit.

VITAL SIGNS: Vital signs (supine systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded.

Laboratory Analyses:

Blood sample will be collected for the following evaluations:

-   -   Hematology: hemoglobin, complete blood count with differential,        INR, PT, and activated PTT    -   Chemistry: electrolytes, creatinine and blood urea nitrogen,        bilirubin, alkaline phosphatase, ALT and AST, calcium, total        protein and albumin    -   Antibody to the Ad-5 virus sample.

Laboratory samples drawn in response to a clinically significant eventwill be documented as unscheduled laboratory evaluations.

Urine sample will be collected for the following evaluations:

-   -   Routine urinalysis    -   Dipstick for Proteinuria    -   No dose modifications for grade 1/2 events    -   Grade 3-(UPC>3.5, urine collection >3.5 g/24 hr, or dipstick        4+): Hold bevacizumab treatment until ≦Grade 2, as determined by        either UPC ratio ≦3.5 or 24 hr collection ≦3.5 g    -   Grade 4 (nephrotic syndrome): Discontinue bevacizumab        Study Therapy Termination: Combination Treatment with VB-111 and        Bevacizumab

Patients will continue to receive VB-111 every 8 weeks and bevacizumabevery 2 weeks, provided that they continue to be stable. Visits willcontinue on days of combination treatment (every 8 weeks). Once patientsexperience disease progression, they will return to the clinic for afinal study visit (see below). In addition, if patients withdraw consentor it is determined by the Investigator that the patient should notcontinue in the study, the patients will return for a final study visit.Patients will be contacted every 2-3 months to follow up on survival.

Follow up will continue until the patient expires.

Bevacizumab Administration as Monotherapy

Bi-Weekly Treatment with Bevacizumab (Days 14, 28, 42 . . . 70, 84, 98,Etc.)

BEVACIZUMAB ADMINISTRATION: Bevacizumab will be administered biweekly byinfusion at a dose of 10 mg/kg according to standard of care practices.During the visits to the clinic to receive this treatment, thesubject/patient should be seen by the Investigator to assess safety.

ADVERSE EVENTS: Patients will be asked about changes in their physicaland mental status since signing the consent form.

CONCOMITANT MEDICATIONS: Patients will be asked about changes in theirtherapies since signing the consent form.

Bevacizumab Treatment (Days 56, 112, 168, etc.)

The following assessments will be performed:

ADVERSE EVENTS: Patients will be asked about changes in their physicaland mental status since the last visit.

CONCOMITANT MEDICATIONS: Patients will be asked about changes in theirtherapies since the last visit.

HEALTH-RELATED QUALITY OF LIFE: EORTC core Quality of Life Questionnaire(QLQ-C30) and a Brain Cancer Module (BCM20) will be completed at eachvisit prior to receiving treatment.

TUMOR MEASURE: Patients will be assessed for response using contrast andnon-contrast brain magnetic resonance imaging (MRI) with assessmentbased on the RANO criteria. MRI imaging will be done up to 72 hoursprior to dosing to assess extent of cancer.

PHYSICAL EXAM: Physical Examination will focus on tumor organs and tumormeasurements, according to RANO criteria, as well as the following: Headand neck; Eyes; Lungs; Heart; Abdomen; Joints; Peripheral circulation;Skin; or Neurologic.

ECG (to be performed 6 months from the initial bevacizumab dose): Astandard 12-lead electrocardiogram with rhythm strip should beperformed.

LABORATORY ANALYSES (according to the operation manual):

Blood sample will be collected for the following evaluations:

-   -   Hematology: hemoglobin, complete blood count with differential,        INR, PT and activated PTT;    -   Chemistry: electrolytes, creatinine and blood urea nitrogen,        bilirubin, alkaline phosphatase, ALT and AST, calcium, total        protein and albumin;    -   Serum pregnancy test    -   Antibody to the Ad-5 virus sample.

Urine will be collected for the following evaluations:

-   -   Routine urine analysis    -   Dipstick for proteinuria        -   No dose modifications for grade 1/2 events        -   Grade 3-(UPC>3.5, urine collection >3.5 g/24 hr, or dipstick            4+): Hold bevacizumab treatment until <Grade 2, as            determined by either UPC ratio ≦3.5 or 24 hr collection ≦3.5            g        -   Grade 4 (nephrotic syndrome): Discontinue bevacizumab

VITAL SIGNS: Vital signs (supine, systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded 15 minutes prior to dosing.

BEVACIZUMAB ADMINISTRATION: Bevacizumab will be administered by infusionat a dose of 10 mg/kg before VB-111 on dosing days. The rate of infusionshall be according to the package insert for bevacizumab: The initialbevacizumab dose should be delivered over 60 minutes (+30 minute window)as an IV infusion.

Study Completion/Early Termination: Monotherapy with Bevacizumab:

Patients will continue to receive bevacizumab every 2 weeks, providedthat they continue to be stable. Visits will continue every 8 weeks.Once patients experience disease progression, they will return to theclinic for a final study visit (see Section 4.5). In addition, ifpatients withdraw consent or it is determined by the Investigator thatthe patient should not continue in the study, the patient will returnfor a final visit.

Patients will be contacted every 2-3 months to follow up on survival.

Follow up will continue until the patient expires.

Study Therapy Termination (All Therapies)

At the time the subject/patient has been withdrawn from the trial, dueto consent withdrawal or disease progression the following assessmentswill be performed as a final study visit:

ADVERSE EVENTS: Patients will be asked about changes in their physicaland mental status since the last visit.

CONCOMITANT MEDICATIONS: Patients will be asked about changes in theirtherapies since the last visit.

HEALTH-RELATED QUALITY OF LIFE AND NEUROCOGNITIVE FUNCTION: EORTC coreQuality of Life Questionnaire (QLQ-C30) and a Brain Cancer Module(BCM20) will be completed.

TUMOR MEASURE Patients will be assessed for response using contrast andnon-contrast brain magnetic resonance imaging (MRI) with assessmentbased on the RANO criteria.

PHYSICAL EXAM: Physical Examination will focus on tumor organs and tumormeasurements, according to RANO criteria, as well as the following: Headand neck; Eyes; Lungs; Heart; Abdomen; Joints; Peripheral circulation;Skin; or Neurologic

ECG: A standard 12-lead electrocardiogram with rhythm strip should beperformed.

Laboratory Analyses:

Blood sample will be collected for the following evaluations:

-   -   Hematology: hemoglobin, complete blood count with differential,        INR, PT and activated PTT    -   Chemistry: electrolytes, creatinine and blood urea nitrogen,        bilirubin, alkaline phosphatase, ALT and AST, calcium, total        protein and albumin;    -   Serum pregnancy test    -   Antibody to the Ad-5 virus sample.

Urine will be collected for the following evaluations:

-   -   Routine urine analysis    -   Dipstick for Proteinuria

VITAL SIGNS: Vital signs (supine, systolic and diastolic blood pressure,peripheral heart rate, body temperature, respiration rate) will berecorded.

Duration of Study

The patients will remain in the study until discontinuation due todisease progression or early termination. Thereafter, the patient willbe contacted by telephone survival data every 2-3 months until thepatient expires.

Correlative/Special Studies Antibodies

Serum samples will be collected for analysis of levels of antibodies tothe adenovirus.

Measurement of Effect Overall Survival

Overall survival is defined as the time from first dose of studymedication until death from any cause. Patients will be followed forsurvival status after completion or removal from the study forprogression or toxicity.

Tumor measurement: Tumor response will be assessed using the samecriteria described in Example 2.

Guidelines for Evaluation of Measurable Disease: The Guidelinesdescribed in Example 2 will be followed.

Response Criteria: Response criteria will be assessed using the samecriteria described in Example 2.

Confirmatory Measurement/Duration of Response

Confirmation: To be assigned a status of PR or CR, changes in tumormeasurements must be confirmed by repeat assessments that should beperformed 4 weeks after the criteria for response are first met.

Duration of Overall Response: The duration of overall response ismeasured from the time measurement criteria are met for CR or PR(whichever is first recorded) until the first date that recurrent orprogressive disease is objectively documented (taking as reference forprogressive disease the smallest measurements recorded since thetreatment started).

The duration of overall CR is measured from the time measurementcriteria are first met for CR until the first date that recurrentdisease is objectively documented.

Duration of Stable Disease: Stable disease is measured from the start ofthe treatment until the criteria for progression are met, taking asreference the smallest measurements recorded since the treatmentstarted.

Exploratory Assessments

Optional Biopsy Samples: Biopsies of brain tissue (preferably freshfrozen samples) may be collected for further testing by VBL as part of aclinically indicated procedure at any time in the study. If a sample ofbrain tissue is collected, a blood sample for transgene analysis shouldbe collected on the same day.

Statistical Considerations Study Design

Objectives: To evaluate the safety, tolerability and efficacy ofmultiple doses of VB-111 1×10¹³ viral particles [VPs] Q2 months asmonotherapy, combined with bevacizumab 10 mg/Kg every 2 weeks uponprogression compared to bevacizumab monotherapy in patients withrecurrent GBM.

All data collected will be summarized and presented. Continuousvariables will be described as the mean, median, standard deviation andrange of n observations. Categorical data will be described withcontingency tables including frequency and percentage. Individualsubject/patient listings will be generated and presented.

All patients meeting the eligibility criteria who signed a consent formand have received VB-111 and/or bevacizumab will be considered evaluablefor safety and efficacy analysis.

Statistical descriptions and analyses will be carried out using SASstatistical analysis software (SAS Institute, Inc., Cary, N.C., USA).

Study Endpoints

Primary Efficacy Endpoint: Overall Survival (OS), is defined as the timefrom first dose of study medication until death from any cause. Patientswill be followed for survival status after completion or removal fromthe study for progression or toxicity.

Secondary Efficacy Endpoints: Progression Free Survival (PFS) is definedas the time from first dose of study medication until objective tumorprogression, assessed according to RANO. Tumor response is measured byRANO Criteria including complete response, partial response, stabledisease and progression.

Tertiary Efficacy Endpoints: Tumor growth rate is defined by the rate ofchange (slope) of tumor size in each subject/patient. Tumor size will bedetermined as the product of the perpendicular dimensions of the tumor.

Health-Related Quality of Life and Neurocognitive Function [EORTC coreQuality of Life Questionnaire (QLQ-C30) and Brain Cancer Module (BCM20)]

Safety and Tolerability Endpoints: adverse events, vital signs, physicalexaminations, laboratory evaluations, and electrocardiogram (ECG).

Statistical Methods

All data collected will be summarized and presented. Continuousvariables will be described as the mean, median, standard deviation,minimum and maximum. Categorical data will be described with contingencytables including frequency and percentage. Individual subject/patientlistings of all data will be generated and presented.

Statistical descriptions and analyses will be carried out using SASstatistical analysis software (SAS Institute, Inc., Cary, N.C., USA).

Study Populations

The intention to treat population (ITT) will include all patientsmeeting the eligibility criteria who have signed a consent form and havereceived VB-111 will be considered evaluable for safety and efficacyanalyses.

In one aspect, efficacy analyses will also be completed in aPer-Protocol population. These analyses will be exploratory and will bedefined in the Statistical Analysis Plan.

In another aspect, all patients who received at least one repeat dose of1×10¹³ VPs of VB-111 will be considered evaluable and included in allefficacy analyses.

Demographic and Baseline Parameters

Demographic and baseline parameters including sex, age, height andweight, details of initial diagnosis and treatment history will besummarized overall and by treatment group. Stratification parametersincluding age (≦60 years, >60 years), KPS (<80, ≧80), and progressionhistory (1^(st) progression, 2^(nd) progression) will be summarizedoverall by treatment group. All continuous variables will be summarizedby descriptive statistics. All discrete variables will be summarized byfrequencies and percentages.

Study Duration and Compliance

All study drug administration and compliance data will be summarized.

Prior and Concomitant Medication

All relevant prior medication and all concomitant medications will besummarized by frequencies and percentages. All medications will be codedusing the World Health Organization (WHO) drug dictionary.

Efficacy Analyses Primary Efficacy Analysis

Overall survival rates including median survival time and 12 monthsurvival rate will be estimated using the Kaplan-Meier method. OS willbe measured from the time of the first dose until death or until thelast known date on which the patient was alive. Patients will befollowed for survival status after completion or removal from the studyfor progression or toxicity until death. For patients who have not diedat the time of analysis, survival data will be censored at the patient'slast known alive date. The log rank test will be used to compare thesurvival distributions of the two treatment groups. In another aspect,the Cox Regression model with the stratification variables as adjustingcovariates will be used to compare the survival distributions of the twotreatment groups.

Secondary Efficacy Analyses

Progression free survival rates will be estimated using the Kaplan-Meirmethod. PFS will be measured from the time of the first dose to the dateof first progression or death or until the last known date on which thepatient was stable. Patients will be followed for progression statusafter completion of the study. The log rank test will be used to comparethe survival distributions of the two treatment groups. In anotheraspect, the Cox Regression model with the stratification variables asadjusting covariates will be used to compare the survival distributionsof the two treatment groups.

Tumor response will be assessed every 2 months until diseaseprogression. The proportion of patients who had complete response,partial response, stable disease and progression will be summarized ateach visit by treatment group. Differences between the proportions ineach group will be tested using a Chi Square test. In another aspect,differences between the proportions in each group will be tested using aCochran Mantel Haenszel (CMH) test controlling for age, KPS, andprogression history.

Tertiary Efficacy Analyses

Tumor measurements will be taken every 2 months until diseaseprogression. Tumor growth rate will be determined by calculating thechange and percent change from baseline of the tumor size at each visit.The rate of change (slope) of each patient will be calculated andsummarized by treatment group. The difference in log transformed tumorgrowth rate will be compared between the treatment group using the 2sample T test or Wilcoxon non parametric test, as appropriate.

Subgroup Analyses

Subgroup analysis for the primary and secondary efficacy endpoints willbe performed for the following categories:

-   -   Age (≦60, >60);    -   KPR (<80, ≧80);    -   First or second disease progression;    -   Residual tumor size post the initial surgical resection (cutoff        will be determined prior to study unblinding);    -   Tumor size at study enrollment (cutoff will be determined prior        to study unblinding);    -   MGMT mutation status;    -   Gender    -   Country

The detailed definitions of the subgroup analyses will be outlined inthe statistical analysis plan. The approach to inference will be aconservative one, based on interactions between treatment group andsubgroup category, and adjusting for multiple comparisons.

Safety and Toxicity Analyses

Adverse events monitoring and clinical findings including physicalexaminations, vital signs, laboratory test results, concomitantmedications, and withdrawals/terminations will be used to assess safety.

Adverse Events

AEs will be categorized by SOC and Preferred Terms using the MedDRAdictionary. The incidence of AEs as well as the severity andrelationship to study drug will be presented by treatment group. Theincidence of AEs leading to withdrawal from the study and serious AEs(SAES) will be summarized by frequency and percentages.

As per NCI CTCAE Version 4.0, the term toxicity is defined as adverseevents that are classified as either possibly, probably, or definitelyrelated to study treatment. The maximum grade for each type of toxicitywill be recorded for each patient and will be used for reporting.Frequency tables will be reviewed to determine toxicity patterns. Inaddition, all adverse event data graded as 3, 4, or 5 and classified aseither “unrelated or unlikely to be related” to study treatment in theevent of an actual relationship developing will be reviewed.

Serious adverse events will be similarly summarized.

Infusional adverse events will also be summarized by both theinvestigator's and the sponsor's assessment of causality.

Clinical findings will include evaluation of physical examinations,vital signs and laboratory test results, concomitant medications, andwithdrawals/terminations. These findings will be summarized by treatmentgroup. Continuous variables will be described as the mean, median,standard deviation, and range of n observations. Categorical data willbe described with contingency tables including frequency and percent.

Interim Analysis

A Data and Safety Monitoring Committee (DSMC) will be appointed toreview the data on safety and efficacy as they accumulate.

Data on adverse outcomes will be reviewed on an ongoing basis. Thecommittee will meet at regular intervals to be agreed upon by themembers of the committee and the sponsor.

The final analysis of the results will be conducted after all patientshave been followed for at least 12 months. A single formal interimanalysis will be conducted 8 months before the planned final analysis,when it is calculated that 60% of the expected total number of eventswill have accrued. The statistical guideline for the committee torecommend early termination of the trial due to efficacy/futility willbe based on attainment of a statistically significant difference in thedirection of benefit to VBL-111 at the two-sided 0.003 level.

Under the stopping rule, computer simulations show that in order topreserve the overall two-sided 0.05 level, the final analysis should usea two-sided 0.0493 level instead of 0.05.

In another aspect, the statistical guideline for the committee torecommend early termination of the trial due to efficacy/futility willbe based on the conditional probability (under the alternativehypothesis) of attaining a statistically significant result at the endof the trial being less than 0.05. Under this futility stopping rule,the significance level of the final analysis will remain at thetwo-sided level of 0.05, because futility analyses do not increase theprobability of a type I error in the direction of advantage to theexperimental treatment.

At the time of this interim analysis, the Data and Safety MonitoringCommittee will provide the sponsor with the estimated Kaplan-Meirsurvival curves for the two treatment groups together with the estimatedhazard ratio and two-sided p-value for the comparison.

Power and Significance Level

The trial is planned to enroll 252 subjects/patients, who will berandomized to VB-111 vs. bevacizumab, at a 1:1 ratio. See FIG. 1. Thefinal analysis of the results will be conducted after all patients havebeen followed for at least 12 months. The expected number of deaths thatwill have been observed at that time is 151, under the assumption thatrecruitment of all patients will be achieved at a uniform rate over 12months. If recruitment takes longer than 12 moths or recruitment rates,instead of being uniform, increase as the trial proceeds, then theexpected number of deaths will be larger than 151. The sample sizejustification is described below:

Assuming a proportional hazards model, the hazard ratio is calculated asthe ratio of the logarithms of the survival rates (at any time).Assuming a 37.6% 12 month survival rate in the bevacizumab treatmentgroup (based on bevacizumab study) and a 55% 12 month survival rate inthe VB-111 group (based on the 12 patients treated at high dose ofVB-111 reported above), the hazard ratio λ=ln(0.55)/ln(0.376)=0.611.

It is expected that this study will recruit patients for 12 months andthen follow up for 12 months.

A single formal interim analysis will be conducted after 60% of theexpected total number of events will have accrued. In order to preservean overall two-sided 0.05 significance level, the final analysis willuse a two-sided 0.0493 level instead of 0.05. Based on the bevacizumabstudy and, where necessary, on exponential decay extrapolations, thepercentage of deaths observed in the bevacizumab group is expected to be75.4%. Given the hazard ratio of 0.611, the percentage of deathsobserved in the VB-111 group is expected to be 58%.

The final analysis will compare the survival curves of bevacizumabversus VB-111 using the Logrank Test. In order to calculate the requiredsample size, the following formula was used:

$\begin{matrix}{{\frac{1}{d_{1}} + \frac{1}{d_{2}}} = \frac{( {\ln \; \lambda} )^{2}}{( {z_{\alpha} + z_{\beta}} )^{2}}} & (1)\end{matrix}$

whered₁=number of deaths observed in VB-111 groupd₂=number of deaths observed in the bevacizumab groupλ=assumed hazard ratio=0.611z_(α)=standard normal deviate for two-sided significance level of 4.93%(adjusted for the interim analysis to preserve the overall 5%level)=1.966z_(β)=standard normal deviate for statistical power of 85%=1.0364 Given:d₁=p₁×n₁, where n₁ is the sample size in the VB-111 group;d₂=p₂×n₂, where n₂ is the sample size in the bevacizumab group;p₁=overall proportion of deaths expected to be observed in the VB-111group=0.58;p₂=overall proportion of deaths expected to be observed in thebevacizumab group=0.754; and n₁=n₂Solve formula (1) for n₁ and n₂.These numbers were divided by 0.9 to account for a 10% drop-out rate.

Based on the assumptions above, a sample size of 126 patients in thebevacizumab group and 126 patients in the VB-111 group, giving a totalof 252 patients will be required. (FIG. 1).

VB-111 Infusion Preparation

Drug Description

Study drug vials will be supplied for each patient in labeled 1.8 mlcryovials (polypropylene) or 10 ml borosilicate vials. Each cryovialcontains a volume of 1.1 ml (1012 VP/ml). Each borosilicate vialcontains a volume of 5.3 ml (1012 VP/ml). The drug will be stored in thepharmacy at or below −65° C. The patient information (Patient number,Visit Date) should be added by hand to the label using a permanent inkmarker and recorded in the patient records.

Procedure Prior to Infusion

The entire process of drug preparation shall be carried out at roomtemperature in the BSC type II room. After thawing, the drug may bemaintained up to 3 hours in ice water during preparations. The drug isdiluted in room temperature saline. The preparation of the drug and druginjection shall be completed within 1 hour, with a window of 30 minutes(for a maximum time of 90 minutes).

The pharmacist preparing the drug shall verify that the information onthe container is appropriate for the study and for the patient: productname, concentration, batch number.

For each patient, use the appropriate syringe as specified in Table 9.Place volume needed of saline (brought to room temperature) in a 50 mlsterile plastic tube. Thaw the vials of VB-111 solution by rubbingbetween the gloved hands. Be sure to mark the time of thaw.

Using a 10 ml syringe, pull 1 ml of VB-111 from each of the cryovial or5 ml from the borosilicate vial intended for the specific patient. AddVB-111 to the plastic tube containing the saline solution prepared inadvance. Draw the piston to mix the remaining VB-111 in the syringe withsaline and push it back into the plastic tube. Mix the diluted drug byswirling the contents by hand. Determine the volume to be appliedaccording to the patient's weight and draw the required volume forinjection into the syringe for administration.

After completing the preparation, perform a reconciliation process:check that the correct number of source vials was used; and record vialsassigned to the patient in the drug accountability log.

After preparation of the drug solution, clean the drug formulation areain the pharmacy according to the pharmacy procedures.

VB-111 Infusion

Patients should receive VB-111 in a fasting state. “A fasting state”means that the patient has had nothing to eat or drink for at least theprevious 2 hours.

A single intravenous infusion of the diluted VB-111 should beadministered according to the instructions below at 3 ml/minute.

An infusion pump may be used. A regular meal may be provided to thepatient 30 minutes after completion of dosing. On dosing days where thepatient is treated with both VB-111 and bevacizumab, bevacizumab shallbe prepared and dosed prior to VB-111.

TABLE 9 VB-111 Preparation Dose 1 × 10¹³ 1 × 10¹³ Concentration in vial(VP/ml) 10¹² 10¹² Volume of VB-111 in tube 1.1 ml  5.3 ml  # Vials ofVB-111 10 2 Take this volume of VB-111 10 × 1 ml   2 × 5 ml   SyringeType for VB-111 10 ml 10 ml Volume of saline 40 ml 40 ml Tube type forsaline  50 ml*  50 ml* Total volume prepared 50 ml 50 ml Volume toinject patient weight Entire volume (50 ml) Entire volume ≧50 kg (50 ml)Volume to inject patient weight  35 ml**  35** <50 kg *The pharmacy mayalternatively use a sterile empty bag and add 40 ml NS + 10 ml VB-111 tothe bag, or the pharmacy can use a 50 ml bag of NS and remove the excessvolume then add the VB-111. Either way is an acceptable pharmacypractice. **35 ml for patients <50 kg represents a 30% reduction ofVB-111 mixed with saline, in accordance with the Protocol, “Study DrugAdministration.”

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The present application claims priority to U.S. provisional ApplicationNo. 61/760,601, filed Feb. 4, 2013, U.S. provisional Application No.61/800,716, filed Mar. 15, 2013; U.S. provisional Application No.61/858,467, filed Jul. 25, 2013; and U.S. provisional Application No.61/907,286, filed Nov. 21, 2013, all of which are incorporated herein byreference in their entireties.

1.-51. (canceled)
 52. A method of reducing the size, slowing the growth,or stopping the growth of a tumor in a subject in need thereofcomprising: (i) administering to the subject a vector which comprises aFas-chimera gene operably linked to an endothelial cell-specificpromoter, wherein the responsiveness of the subject to a VEGF antagonistis increased after the administration of the vector, and (ii)administering a VEGF antagonist to the subject, wherein the tumor sizeor tumor growth in the subject is reduced after administration of theVEGF antagonist.
 53. The method of claim 52, wherein the VEGF antagonistis an anti-VEGF antibody or a VEGF binding molecule.
 54. The method ofclaim 53, wherein the anti-VEGF antibody is a monoclonal antibody, ahumanized antibody, a human antibody, a single chain antibody, or achimeric antibody.
 55. The method of claim 54, wherein the anti-VEGFantibody is bevacizumab.
 56. The method of claim 52, wherein the vectoris administered at an effective amount of about 1×10¹¹ virus particlesto about 1×10¹⁴ virus particles.
 57. The method of claim 55, wherein thebevacizumab is administered at an effective amount of about 1 mg/kg toabout 15 mg/kg.
 58. The method of claim 55, wherein the vector isadministered at an, effective amount of about 1×10^(12i) to about 1×10¹³virus particles and bevacizumab is administered at an effective amountof 5 mg/kg to 15 mg/kg.
 59. The method of claim 53, wherein the VEGFantagonist is selected from the group consisting of bevacizumab,ranibizumab, VGX-100, r84, aflibercept, IMC-18F1, IMC-1C11, andramucirumab.
 60. The method of claim 53, wherein the vector isrepeatedly administered.
 61. The method of claim 60, wherein the vectoris repeatedly administered once in about 2 weeks to once in about 6months.
 62. The method of claim 55, wherein the bevacizumab isrepeatedly administered.
 63. The method of claim 62, wherein thebevacizumab is repeatedly administered once in about 7 days to once inabout 6 months.
 64. The method of claim 55, wherein the vector isrepeatedly administered every 2 months and the bevacizumab is repeatedlyadministered every 2 weeks.
 65. The method of claim 52, wherein thetumor is a metastatic tumor.
 66. The method of claim 52, wherein thevector comprises, consists of, or consists essentially of the nucleotidesequence set forth in SEQ ID NO:
 19. 67. A method of treating a diseaseor condition associated with tumor in a subject in need thereofcomprising: (i) administering to the subject a vector which comprises aFas-chimera gene operably linked to an endothelial cell-specificpromoter, wherein the responsiveness of the subject to a VEGF antagonistis increased after the administration of the vector, and (ii)administering a VEGF antagonist to the subject, wherein the disease orcondition in the subject is treated after administration of the VEGFantagonist.
 68. The method of claim 67, wherein the tumor of the subjectis progressed after administration of the vector.
 69. The method ofclaim 67, wherein the vector comprises, consists of, or consistsessentially of the nucleotide sequence set forth in SEQ ID NO:
 19. 70.The method of claim 67, wherein the VEGF antagonist is selected from thegroup consisting of bevacizumab, ranibizumab, VGX-100, r84, aflibercept,IMC-18F1, IMC-1C11, and ramucirumab.
 71. The method of claim 67, whereinthe tumor is associated with Leukemia, Hodgkin's Disease, Non-Hodgkin'sLymphoma, multiple myeloma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid, urinarybladder cancer, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, papillary thyroid cancer, neuroblastoma, neuroendocrinecancer, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, cervical cancer, endometrial cancer, adrenal corticalcancer, prostate cancer, Mullerian cancer, ovarian cancer, peritonealcancer, fallopian tube cancer, uterine papillary serous carcinoma,metastatic colorectal cancer (mCRC), advanced nonsquamous non-small celllung cancer (NSCLC), metastatic renal cell carcinoma (mRCC), orglioblastoma multiforme (GBM).